Table of Contents T ABLE OF C ONTENTS CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 CHAPTER 5 CHAPTER 6 CHAPTER 7 CHAPTER 8 CHAPTER 9 CHAPTER 10 CHAPTER 11

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1 T ABLE OF C ONTENTS Table of Contents CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 CHAPTER 5 CHAPTER 6 CHAPTER 7 CHAPTER 8 CHAPTER 9 CHAPTER 10 CHAPTER 11 CHAPTER 12 APPENDIX A The Basics of PCR and RT-PCR The Basics of PCR The Basics of RT-PCR Increasing RT-PCR Sensitivity Isolating High-Quality RNA Using RNase H Reverse Transcriptases Higher RT Incubation Temperatures Additives to Enhance RT RNase H Treatment Improving Detection of Small Amounts of RNA One-Step vs. Two-Step RT-PCR Improving RT-PCR Specificity Priming cdna Synthesis Higher RT Incubation Temperatures Minimizing Contaminating Genomic DNA RT-PCR Applications and 3 RACE Quantifying mrna Expression Improving PCR Specificity Primer Design Primer Annealing Temperature Touchdown PCR Primer Concentration Primer Purity and Stability Hot Start Magnesium Concentration Additives to Enhance PCR Nested PCR Increasing PCR Sensitivity Template Quality Template Concentration Enzyme Choice Improving Fidelity Enzymes with Proofreading Enzyme Mixes Other Parameters Other Things to Consider Amplifying Long Targets Prevention of Carry-Over Contamination Purification of PCR Products PCR Applications Multiplex PCR Genotyping with Dinucleotide Repeat Markers Tools for Detecting Polymorphisms Troubleshooting Guide References Related Products Selected Primer Sequences

2 C HAPTER 1 The Basics of PCR and RT-PCR The Basics of PCR The Polymerase Chain Reaction (PCR) process uses multiple cycles of template denaturation, primer annealing, and primer elongation to amplify DNA sequences (1). It is an exponential process since amplified products from the previous cycle serve as templates for the next cycle of Component Template Table 1 Final Concentration copies of DNA template Primer µm Primer µm 10X Reaction buffer Magnesium dntp mix Thermostable DNA polymerase TABLE 1. Reaction components. amplification, making it a highly sensitive technique for the detection of nucleic acids. Typically, enough amplified product is generated after 20 to 30 cycles of PCR so that it can be visualized on an ethidium bromide-stained gel. The reaction is composed of several components (table 1). The template can include purified genomic or plasmid DNA; RNA converted to cdna; or unpurified, crude biological samples such as bacterial colonies or phage plaques. The primers determine the sequence and the length of the amplified product. The most frequently used thermostable polymerase is Taq DNA polymerase. This enzyme is appropriate for routine amplifications, but the use of other thermostable polymerases can enhance results. Amplification reactions also contain buffer, deoxynucleotide triphosphates, and magnesium. The magnesium ion concentration affects enzyme activity, primer annealing, melting temperature of the template and the PCR product, fidelity, and primer-dimer formation (2). How the interaction of these components, cycling parameters, as well as other factors contribute to successful PCR will be discussed in the following chapters. 1X mm 200 µm each dntp 1 4 units/100 µl reaction The Basics of RT-PCR RT-PCR combines cdna synthesis from RNA templates with PCR (figure 1) to provide a rapid, sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify the expression of messages, often from small amounts of RNA (3,4). In addition, the technique is used to analyze differential gene expression or clone cdnas without constructing a cdna library (5,6). RT-PCR is more sensitive and easier to perform than other RNA analysis techniques, including Northern blots, RNase protection assays, in situ hybridization, and S1 nuclease assays (7,8). The template for RT-PCR can be total RNA or poly(a) + selected RNA. RT reactions can be primed with random primers, oligo(dt), or a gene-specific primer (GSP) using a reverse transcriptase. RT-PCR can be carried out either in two-step or one-step gene specific priming GSP FIGURE 1. RT-PCR overview. AAAAAAA(A)n AAAAAAA(A)n AAAAAAA(A)n Cells or Tissue RNA Isolation formats. In two-step RT-PCR, each step is performed under optimal conditions. cdna synthesis is performed first in RT buffer and one tenth of the reaction is removed for PCR. In one-step RT-PCR, reverse transcription and PCR take place sequentially in a single tube under conditions optimized for both RT and PCR. This Guide describes the keys to successful RT-PCR and PCR. High sensitivity (getting enough product from small samples) and high specificity (selective amplification of only the desired product) are the hallmarks of successful PCR. Optimal RT-PCR and PCR can be obtained through careful experimental design, including selecting the appropriate enzymes, designing optimal primers, using different buffers and additives, establishing cycling parameters, and preparing high quality templates. AAAAAAA(A)n AAAAAAA(A)n cdna Synthesis oligo(dt) priming AAAAAAA(A)n TTTTTTTT AAAAAAA(A)n AAAAAAA(A)n TTTTTTTT AAAAAAA(A)n TTTTTTTT PCR Amplification N6 random hexamer priming N6 N6 N6 N6 N6 N6 N6 N6 AAAAAAA(A)n N6 AAAAAAA(A)n AAAAAAA(A)n 2 THE BASICS OF PCR AND RT-PCR

3 C HAPTER 2 Increasing RT-PCR Sensitivity Isolating High-Quality RNA Successful cdna synthesis starts with high-quality RNA. High-quality RNA is substantially full length, and does not contain inhibitors of reverse transcriptases such as EDTA or SDS (9). The quality of the RNA dictates the maximum amount of sequence information that can be converted into cdna. One popular RNA isolation protocol is the single-step method which uses guanidine isothiocyanate/acidic phenol (10). The TRIZOL Reagent method (see figure below) is an improvement on this single-step method and can be used to isolate high quality, undegraded RNA from various cells and tissues (11,12). The TRIZOL Reagent method can be used to isolate RNA from as little as 100 cells or 1 mg of tissue (13). Oligo(dT) selection for poly(a) + RNA is typically not necessary. Amplified product is detectable when either total RNA or poly(a) + RNA is used as the starting template (figure 2) (14). In addition, isolating poly(a) + RNA may lead to variability of mrna enrichment between samples, which can bias the detection or quantification of message. However, poly(a) + RNA may increase the sensitivity of detection when analyzing rare messages. RNA isolated from RNase-rich samples, such as the pancreas, may need to be stored in formamide to maintain high quality RNA, due to the carry-over of trace amounts of RNase (15). This is especially true for long term storage. RNA isolated from rat pancreas and stored in water showed substantial degradation after just 1week, whereas RNA isolated from rat liver, which contains lower amounts of RNases, was stable in water for 3 years (15). In addition, transcripts >4 kb are more TRIZOL Reagent can isolate high-quality RNA from as little as 100 cells or 1 mg of tissue. susceptible to degradation by trace amounts of RNases than shorter transcripts (16). To increase the stability of stored RNA samples, dissolve the RNA in deionized formamide and store at 70 C. The formamide used for storage of RNA must not contain impurities that cause RNA degradation (16). RNA from pancreas was stable for at least 1 year in formamide. When ready to use the RNA, simply precipitate it by adding NaCl to 0.2 M followed by 4 volumes of ethanol. Incubate 3 to 5 min at room temperature and centrifuge at 10,000 g for 5 min. Outline of Procedure for TRIzOL Reagent Homogenize sample in TRIZOL Reagent Separate Phases (add chloroform) Precipitate RNA Wash and Solubilize Elapsed Time <1 h RNase inhibitors are often added to RT reactions to help improve cdna length and yield. Add RNase inhibitors to the firststrand reaction in the presence of buffer and a reducing agent, such as DTT, since procedures prior to cdna synthesis that denature the inhibitor will release bound RNases that can degrade RNA. Protein RNase inhibitors protect RNA against degradation by RNases A, B, and C, but do not protect against RNases found on the skin. Thus, even when using these inhibitors be careful not to introduce RNases from your fingertips. 100 bp DNA Ladder Panel A Panel B Total RNA Poly(A) + RNA bp DNA Ladder FIGURE 2. Comparison of total RNA and poly(a) + RNA in RT-PCR. cdna was synthesized in duplicate from 5 or 1 µg total HeLa RNA (lanes 1 and 2, respectively) or from 500 ng or 50 ng of poly(a) + HeLa RNA (lanes 3 and 4, respectively) with oligo(dt) primers and SUPERSCRIPT II RT. The amplified targets were a 377-bp fragment near the 5 end of the DNA polymerase ε mrna and a 643-bp fragment of the replication protein A mrna, which are both moderately abundant targets. One tenth of the cdna reaction was amplified for 30 cycles using Taq DNA polymerase. 377 bp 643 bp INCREASING RT-PCR SENSITIVITY 3

4 C HAPTER 2 Increasing RT-PCR Sensitivity (continued) Using RNase H Reverse Transcriptases Reverse transcriptases catalyze the conversion of RNA to cdna. Both moloney murine leukemia virus (M-MLV) and avian myleoblastosis virus (AMV) reverse transcriptases have endogenous RNase H activity in addition to their polymerase activity. The RNase H activity competes with the polymerase activity for the hybrid formed between the RNA template and the DNA primer or growing cdna strand and degrades the RNA strand of the RNA:DNA complex (see figure). RNA template that is cleaved by RNase H activity is no longer an effective substrate for cdna synthesis, decreasing both the amount and size of the cdna. Therefore, it is advantageous to eliminate or greatly reduce the RNase H activity of reverse transcriptases. cdna Synthesized (ng) AMV RT Figure 3 THERMOSCRIPT RT M-MLV RT FIGURE 3. Effect of RT on yield of first-strand cdna. cdna was synthesized from 2.5 µg (for THERMOSCRIPT RT and AMV) or 1 µg (for SUPERSCRIPT II RT and M-MLV) of a 7.5-kb mrna using oligo(dt) primer and 10 µci of [α- 32 P]dCTP with the recommended synthesis conditions. Total yield of first-strand ( ) was calculated from TCA precipitation. Full-length cdna ( ) was assayed by cutting and counting size-fractionated bands from alkaline agarose gels. SUPERSCRIPT II RT, an M-MLV RNase H RT, and THERMOSCRIPT RT, an avian RNase H reverse transcriptase, yield greater amounts of cdna and more full-length cdna than M-MLV RT and AMV RT, respectively (figure 3) (17 19). RT-PCR sensitivity can be limited by the amount of cdna synthesized. THERMOSCRIPT RT has significantly greater sensitivity than AMV RT (figure 4) (18). The size of RT-PCR products is limited by the ability of a reverse transcriptase to synthesize full-length cdna, especially when cloning large cdnas. SUPERSCRIPT II RT significantly increased the yield of long RT-PCR products compared to M-MLV RT (figure 5) (20). RNase H reverse transcriptases also have increased thermostability, so reactions can be performed above the normal incubation temperature of 37 C to 42 C. SUPERSCRIPT II RT THERMOSCRIPT RT ,000 1 Kb PLUS DNA Ladder AMV RT ,000 FIGURE 4. Effect of RT on sensitivity of RT-PCR. cdna was synthesized from total HeLa RNA at 50 C with oligo(dt) primers using THERMOSCRIPT RT or AMV RT. PCR was 35 cycles with PLATINUM Taq DNA Polymerase and primers for human DNA polymerase ε. FIGURE 5. Effect of RT on sensitivity in long RT-PCR. The synthesis of full-length cdna for human tuberous sclerosis II mrna (5.3 kb) and human DNA polymerase ε mrna (6.8 kb) was catalyzed by SUPERSCRIPT II RT (lane S) or M-MLV RT (lane M). cdna was synthesized from 5 µg total HeLa RNA with oligo(dt) primers. Samples were treated with RNase H and one tenth of the cdna reaction was amplified with ELONGASE Enzyme Mix for 35 cycles. Obstacles Posed by RNase H TTTTTTTT AAAAAAAA A TTTTTTTT AAAA S M S M TTTTTTTT AAAAAAAA TTTTTTTT AAAAAAAA Effect of RNase H on first-strand cdna. RNase H degrades RNA in an RNA:DNA complex during cdna synthesis. Red arrows represent potential cleavage sites. RNA (ng) 3.5 kb kb INCREASING RT-PCR SENSITIVITY

5 C HAPTER kb Higher RT Incubation Temperatures Higher RT incubation temperatures (table 2) can help disrupt RNA secondary structure and increase the yield of product (18). For many RNA templates, incubating the RNA and primer at 65 C in the absence of buffers or salts and quickly chilling on ice eliminates most secondary structure and allows primer binding. However, secondary structure may still exist in some templates even after heat denaturation. The amplification of products from these difficult templates can be improved by using THERMOSCRIPT RT and performing the RT reactions at higher temperature (figure 6) (18). Higher temperatures can also improve specificity, especially when using a gene-specific primer (GSP) for cdna synthesis (see page 7). If using a GSP, make sure the T m of the primer is as high as the planned incubation temperature. Do not use oligo(dt) and random primers above 60 C; random primers require an initial 10 min incubation at 25 C before increasing the temperature to 60 C. In addition to using Reverse Transcriptase AMV Table 2 Incubation Temperature 37 C 45 C M-MLV 37 C SUPERSCRIPT II RT THERMOSCRIPT RT THERMOSCRIPT RT 1 Kb PLUS AMV RT 50 C 55 C 60 C DNA Ladder 50 C 55 C 60 C FIGURE 6. Effect of temperature on a difficult template. cdna was synthesized at the indicated temperatures from 10 ng of soybean total RNA with THERMOSCRIPT RT or AMV RT and a gene-specific primer for 18S rrna. 40 cycles of PCR was performed with one tenth of the cdna reaction and PLATINUM Taq DNA Polymerase High Fidelity. 37 C 50 C 42 C 65 C* *RNA begins to hydrolyze at temperatures above 65 C. For targets 1 kb, 70 C first-stand synthesis temperatures can be used. For targets >1 kb, use first-strand synthesis temperatures 65 C. TABLE 2. RT incubation temperatures. elevated RT temperatures, increase specificity by shifting the RNA/primer mix directly from the 65 C denaturation temperature to the RT incubation temperature and add prewarmed 2X reaction mix (cdna synthesis hot start). This helps prevent intramolecular base pairing that can occur at lower temperatures. Using a thermal cycler can simplify the multiple temperature shifts required for RT-PCR. Tth thermostable polymerase, which functions as a DNA polymerase in the presence of Mg ++ and a reverse transcriptase in the presence of Mn ++, can also be incubated up to 65 C. However, the presence of Mn ++ during PCR reduces fidelity. This makes Tth polymerase less suitable for applications that require highly accurate amplification, such as cloning cdnas. In addition, Tth polymerase is a less efficient reverse transcriptase, which reduces sensitivity and, since a single enzyme is used for both reverse transcription and PCR, a control reaction without RT cannot be used to distinguish amplified cdna from amplified contaminating genomic DNA. Additives to Enhance RT Additives including glycerol and DMSO can be added to first-strand synthesis reactions to help destabilize nucleic acid duplexes and melt RNA secondary structure. Up to 20% glycerol or up to 10% DMSO can be used without effecting SUPERSCRIPT II RT or M-MLV RT activity (9). AMV RT will also tolerate up to 20% glycerol without loss of activity. For maximum RT-PCR sensitivity in SUPERSCRIPT II RT reactions, add 10% glycerol and incubate at 45 C. If one tenth of the RT reaction is added to the PCR, the final concentration of glycerol in the amplification reaction is 0.4%, which will not inhibit PCR. RNase H Treatment Treating cdna reactions with RNase H prior to PCR can improve sensitivity. For some targets, it is thought that the RNA in the cdna reaction may prevent binding of the amplification primers. In these cases, RNase H treatment can improve sensitivity. RNase H treatment is often necessary when amplifying longer, full-length cdna targets, such RNase H S M A + RNase H S M A FIGURE 7. Effect of RNase H treatment on RT-PCR. The synthesis of full-length cdna for human tuberous sclerosis II mrna (5.3 kb) from 5 µg of total HeLa RNA was catalyzed by SUPERSCRIPT II RT (S), M-MLV RT (M), or AMV RT (A). One half of the reaction was treated with RNase H for 30 min. An amount of the treated or untreated RT reaction equivalent to 0.5% of the starting RNA was amplified for 35 cycles with ELONGASE Enzyme Mix. as the low copy target tuberous sclerosis II (figure 7) (20). For this difficult target, RNase H treatment improved the signal from cdna generated with either SUPERSCRIPT II RT or M-MLV RT. For many RT-PCR applications, RNase H treatment is optional since incubation at 95 C for the PCR denaturation step often hydrolyzes the RNA in the RNA:cDNA complex. 5.3 kb INCREASING RT-PCR SENSITIVITY 5

6 C HAPTER 2 Increasing RT-PCR Sensitivity (continued) 500 bp A Improving Detection of Small Amounts of RNA RT-PCR is particularly challenging when only small amounts of RNA are available. The addition of glycogen as a carrier during RNA isolation helps improve the yield from small samples (13). RNase-free glycogen is added at the same time TRIZOL Reagent is added. Glycogen is water soluble and remains in the aqueous layer with the RNA to aid subsequent precipitation. The recommended concentration of RNase-free glycogen is 250 µg/ml for <50 mg of tissue or <10 6 cultured cells. The addition of acetylated BSA (figure 8) to RT reactions performed with SUPERSCRIPT II RT can increase sensitivity (21). Also, for small amounts of RNA, reducing the amount of SUPERSCRIPT II RT and adding 40 units of RNASEOUT Ribonuclease Inhibitor can enhance the level of detection. If glycogen is B FIGURE 8. Effect of BSA on RT-PCR sensitivity. 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, and 0.5 fg (lanes 1 to 6, respectively) of CAT mrna transcript were reverse transcribed without (Panel A) or with (Panel B) 0.5 µg of acetylated BSA using SUPERSCRIPT II RT. The no-rt control contained 50 pg of CAT transcript (lane 7). 30 cycles of PCR was performed with Taq DNA polymerase. TWO-STEP PROCEDURE Prime first-strand cdna with: Oligo(dT) Random hexamers GSP primers Provides: Flexibility Choice of primers. Choice of amplification enzyme. Ability to optimize for difficult RT-PCR. Combine with PLATINUM enzymes for higher specificity. Combine with PLATINUM Pfx DNA Polymerase for greater fidelity. Ideal for detecting or quantifying several messages from a single sample. Table 3 ONE-STEP PROCEDURE Prime first-strand cdna with: GSP primers Provides: Convenience Amplification enzymes premixed with reverse transcriptase. Fewer pipetting steps and reduced chances of contamination. High sensitivity. Ideal for analysis of large numbers of samples. Ideal for quantitative PCR. Notes on amplification enzymes and product size: Use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <4 kb. Use PLATINUM Pfx DNA Polymerase or PLATINUM Taq DNA Polymerase High Fidelity for products <12 kb. Use ELONGASE Enzyme Mix for products >12 kb. For one-step RT-PCR, use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <3.5 kb; and use PLATINUM Taq DNA Polymerase High Fidelity for products <9 kb. used during the RNA isolation, the addition of BSA or RNase inhibitor to SUPERSCRIPT II RT reactions is still recommended. One-Step vs. Two-Step RT-PCR Two-step RT-PCR is popular and useful for detecting multiple messages from a single RNA sample. However, a one-step RT-PCR method offers other benefits (table 3) (22). One-step RT-PCR is easier to use when processing large numbers of samples, and helps minimize carry-over contamination since tubes do not need to be opened between cdna synthesis and amplification. Since the entire cdna sample is amplified, one-step RT-PCR can provide greater sensitivity, down to 0.1 pg total RNA (figure 9) (22). For successful one-step RT-PCR, use an antisense gene-specific primer (GSP) to prime cdna synthesis bp DNA Ladder FIGURE 9. Sensitivity of one-step RT-PCR.A β-actin fragment was amplified from 0, 0.1, 1, 10, 10 2, and 10 3 pg of total HeLa RNA (lanes 1 to 6, respectively) using the SUPERSCRIPT One-Step RT-PCR System. Reactions were incubated at 50 C for 30 min; 94 C for 2 min; then 40 cycles of 94 C for 15 s and 55 C for 30 s and 68 C for 90 s; followed by 68 C for 5 min. Reactions contained 200 nm of sense and antisense primer. 353 bp TABLE 3. Comparison of two-step and one-step RT-PCR. 6 INCREASING RT-PCR SENSITIVITY

7 C HAPTER 3 Improving RT-PCR Specificity Priming cdna Synthesis First-strand cdna synthesis reactions may be primed using three different methods. The relative specificity of each method influences the amount and the variety of the cdna synthesized. Random primers are the least specific of the three methods. The primers anneal to multiple sites along the entire transcript to generate short, partial-length cdnas. This method is often used to capture 5 end sequences and make cdna from RNA templates with regions of secondary structure or pause sites that reverse transcriptases cannot copy (2,3). To maximize the size of the cdna, the ratio of primers to RNA may need to be determined empirically for each RNA preparation. The starting concentration range is 50 to 250 ng of random primers per 20 µl reaction. Since the majority of the cdna synthesized from total RNA with random primers is ribosomal, poly(a) + selected RNA is often used as the template. Oligo(dT) priming is more specific than random primers. It hybridizes to the poly(a) tails found at the 3 ends of most eukaryotic mrnas (23). Since poly(a) + RNA constitutes approximately 1% to 2% of a total RNA population, the amount and complexity of cdna is considerably less than when random primers are used. Because of its higher specificity, oligo(dt) priming generally does not require optimization of the primer-to-rna ratio or poly(a) + selection. The use of 0.5 µg of oligo(dt) primer per 20 µl reaction is recommended. Oligo(dT) is suitable for many RT-PCR applications. Oligo(dT) 20 is provided with the THERMOSCRIPT RT-PCR System, since it has greater thermostability for use at higher RT incubation temperatures. A gene-specific primer (GSP) is the most specific primer for the RT step. GSPs are antisense oligonucleotides that hybridize to specific RNA target sequences, instead of annealing to entire RNA populations as with random primers or oligo(dt). The same rules for designing PCR primers are applied to the design of the GSP for the RT reaction (see chapter 5). A GSP can be the same sequence as the amplification primer which anneals closest to the 3 end of the message; or a GSP can be designed to anneal downstream of the reverse amplification primer. Some targets require the design of more than one antisense GSP for successful RT- PCR, since secondary structure of the RNA target may prevent primer binding. The use of 1 pmole antisense GSP in a 20-µl firststrand reaction is recommended. Higher RT Incubation Temperatures To take full advantage of the specificity provided by GSPs, use an RT with greater thermostability. Thermostable RTs can be incubated at higher temperatures to increase reaction stringency (17,18). For example, if a GSP has an annealing temperature of 55 C, the specificity conferred by the GSP is not fully utilized if the RT reaction is performed with AMV RT or M-MLV RT under low stringency at 37 C. However, SUPERSCRIPT II RT and THERMOSCRIPT RT allow for reaction temperatures of 50 C and greater (table 2, page 5), which can eliminate the nonspecific products generated at lower temperatures (figure 10). When maximizing specificity, add the RNA/primer mix directly from the 65 C denaturation temperature to the RT incubation temperature and add prewarmed 2X reaction mix (cdna synthesis hot start). This helps prevent intramolecular base pairing that can occur at lower temperatures. Using a thermal cycler can simplify the multiple temperature shifts required for RT-PCR. 1 Kb PLUS DNA LADDER 50 C 55 C 60 C 2 kb FIGURE 10. Effect of RT reaction temperature on RT-PCR specificity. cdna was synthesized from 1 µg of HeLa RNA with THERMOSCRIPT RT and a GSP designed to anneal to the human DNA polymerase ε mrna. THERMOSCRIPT RT was added to a prewarmed reaction mixture and 35 cycles of PCR was performed using one tenth of the cdna with PLATINUM Taq DNA Polymerase. Minimizing Contaminating Genomic DNA One potential difficulty encountered with RT-PCR is genomic DNA contamination of RNA. Using a good RNA isolation technique, such as TRIZOL Reagent, minimizes the amount of contaminating genomic DNA in the RNA preparation. To avoid generating products from genomic DNA, remove the contaminating DNA by treating RNA with DNase I, Amplification Grade before reverse transcription. To terminate the DNase I digestion, incubate the sample at 65 C for 10 min in the presence of 2.0 mm EDTA. The EDTA chelates the magnesium and prevents magnesium dependent hydrolysis of the RNA that can occur at higher temperatures. To distinguish amplified cdna from amplified contaminating genomic DNA, design primers that anneal in separate exons. The PCR products derived from cdna will be shorter than products amplified from contaminating genomic DNA. In addition, perform a control experiment without RT for each RNA template to determine whether a given fragment is of genomic DNA or cdna origin. Products generated in the absence of RT are of genomic origin. IMPROVING RT-PCR SPECIFICITY 7

8 C HAPTER 4 RT-PCR Applications 5 and 3 RACE Rapid amplification of cdna Ends (RACE) is a procedure to capture unknown sequences at either the 5 or 3 end of a transcript. Unlike conventional RT-PCR, which employs two sequencespecific primers, RACE uses one sequencespecific primer and either the poly(a) tail of mrnas (3 RACE) or a homopolymeric tail added to cdna ends (5 RACE) (figure 11). RACE has been used for the amplification and cloning of rare mrnas (23-25). RACE products can be cloned, directly sequenced, used to prepare probes, or combined to generate full-length cdna (25-27). One method for joining 5 and 3 RACE products is to use the sequence information generated by 5 and 3 RACE to design new primers which will amplify the entire cdna sequence (26). The use of RNase H RT and high-fidelity thermostable polymerases allows higher fidelity amplifications of longer sequences to generate full-length cdna clones. 5 RACE is more challenging and less specific than RT-PCR applications, since only CC... CC Abridged Anchor Primer 5 GI... 3 CC... IG CC UAP AUAP 5 GI... IG 3 CC... CC mrna Figure 11 GSP2 nested GSP GSP1 FIGURE 11. Summary of the 5 RACE procedure (A) n (A) n Anneal first strand primer, GSP1, to mrna Copy mrna into cdna with SUPERSCRIPT TM II RT Degrade RNA with RNase Mix Purify cdna with GLASSMAX Spin Cartridge Tail purified cdna with dctp and TdT one of the amplification primers is genespecific. 5 RACE products may be a single product, multiple products, or even a smear with no distinguishable products (figure 12) (28). The quality of the result depends on the specificity of the GSPs used in first-strand synthesis and amplification, the specificity of the anchor primer during amplification, the complexity and abundance of the target, and the length of the product. Amplification with nested primers (up to three rounds of nested amplification) and using size-selected amplified product as the target in successive rounds of amplification increases the specificity of 5 RACE (see nested PCR discussion on page 13). Increasing the RT incubation temperature and PCR annealing temperature, as well as decreasing the magnesium concentration in the amplification reaction, can enhance specificity. 5 RACE sensitivity is affected by the RT used and secondary structure at the 3 -end of the cdna that may inhibit cdna tailing. Incomplete cdna synthesis lowers the yield of full-length product and contributes to the smearing pattern observed with some targets since shorter PCR amplify dc-tailed cdna using the Abridged Anchor Primer and nested GSP2. Reamplify primary PCR product using AUAP, or UAP, and nested GSP Kb DNA Ladder kb FIGURE RACE. cdna was synthesized from 5 µg HeLa total RNA with SUPERSCRIPT II RT at 45 C. 10 µl of purified cdna was tailed in the presence of 10% DMSO. 1 µl of the tailing reaction was directly amplified for 40 cycles with primers for human tuberous sclerosis and ELONGASE Enzyme Mix (primary PCR 2.8 kb; lane 1). A 10 µl gel plug was removed around the 2.8-kb band. The DNA was eluted in 50 µl of TE. Nested PCR (30 cycles) was performed using 1 µl of size-selected PCR product (nested PCR 2.7 kb; lane 2). The gel is stained with SYBR Green I. cdnas are tailed and amplified. Since SUPERSCRIPT II RT generates more full-length cdna, it can increase the level of detection of 5 end sequence, especially for transcripts >1 kb (28). Doublestranded 3 termini and hairpin structures impair tailing of cdna by decreasing the availability of the 3 -OH for tailing. The initial incubation of the cdna at 94 C followed by chilling on ice helps to disrupt secondary structure. Some difficult targets may require the addition of DMSO for effective tailing (figure 12) (28). The tailing enzyme, terminal deoxynucleotidyl transferase is tolerant of up to 20% DMSO. If no products are visible or if only a smear is visible after nested amplification, a Southern blot can be used to detect products. This requires internal sequence information to use as a probe. Quantifying mrna Expression Quantifying mrna expression is challenging RT-PCR, but offers advantages over traditional RNA analysis methods. RT-PCR is more sensitive than Northern blot or ribonuclease protection analysis and requires less RNA and sequence information. However, RT-PCR involves two enzymatic steps that can contribute to variability in the amount of RT-PCR product. The amount of RNA converted to cdna affects yield, but the major difficulty in quantitative PCR is the exponential nature of PCR, in which small variations between samples translates to large differences in product yield(29). Two popular 8 RT-PCR APPLICATIONS

9 C HAPTER 4 methods for quantifying mrna abundance are competitive, quantitative PCR and realtime PCR (4,30). In competitive PCR, an exogenous RNA transcript (internal standard RNA) is added before the RT step to control for sample-tosample variation. The internal standard RNA is reverse transcribed and amplified with the target to control for differences in the efficiency of cdna conversion and amplification. Quantitation is performed by coamplification of the specific target sequence together with known concentrations of the internal standard RNA. The abundance of the target is determined by comparing the signal obtained for the internal standard to the signal for the target. The internal standard RNA shares the same primer recognition sites with the target, but is a different size. Several methods exist to generate internal standards (31). One of the simplest methods involves installing the primer recognition sites on nonspecific spacer DNA by PCR (31). This product can be cloned into a vector carrying the T7 or SP6 RNA polymerase promoter, or the RNA promoter sequences can be included on the forward primer in order to install the sequences by PCR (32). Then, RNA standards can be generated by in vitro transcription. Competitive, quantitative PCR can be performed in one-step RT-PCR using a GSP or performed in a two-step reaction using a GSP or oligo(dt). Oligo(dT) sequence can be installed by adding poly(dt) sequences to the reverse primer (32). Competitive RT-PCR is an end-point analysis that requires the amplification efficiency of the target and the internal standard to be equal. Some prior knowledge of the amount of starting target is necessary to establish a concentration range for the internal standard. For accurate quantitation, reactions containing more standard than target and less standard than target are required. Real-time RT-PCR is non-competitive and detects the product as it is being formed. Several probes can be used to assay the accumulation of product including fluorogenic 5 nuclease probes and Molecular Beacons (33,34). The first method is based on the 5 nuclease activity of Taq DNA polymerase, which is used to cleave a hybridization probe at the branch point during extension (33). The assay uses probes with two fluorescent labels, one dye serves as a reporter and the other quenches its signal until the probe is cleaved. Molecular Beacons contain a 5 fluorophore and 3 quencher. These probes are designed with a hairpin structure bringing the fluorophore and quencher in close proximity to quench fluorescence (34). The stem structure is composed of 5 to 7 nucleotides and is GC rich. The outer loop structure is composed of 15 to 30 nucleotides and is complementary to the target sequence. When the target sequence is present, the molecular beacon hybridizes to the target, relaxing the hairpin structure and allowing fluorescence. For both probes, fluorescent measurements are made in real time during each cycle of PCR using a thermal cycler coupled to an optical excitation and detection device. The amount of fluorescence released is proportional to the amount of PCR product. The cycle at which the amount of released fluorescence crosses an established baseline fluorescence is known as the threshold cycle or C T. The C T value is inversely proportional to the amount of starting target. Thus, high-copynumber samples will have low C T values and low-copy-number samples will have high C T values (figure 13). Relative Fluorescence Threshold Cycle (CT) Panel A 2,000 1,800 1,600 1,400 1,200 1, For absolute quantification of messages, a standard curve is generated using an in vitro transcribed RNA. The C T value is determined for different concentrations of the in vitro transcribed RNA. Then the C T values are plotted against the RNA concentrations to generate a standard curve for quantifying the level of expression in unknown samples (figure 13, panel B). Real-time RT-PCR offers advantages over competitive RT-PCR. Fluorescence monitoring is highly sensitive and highly accurate with a linear-dose response over a wide range of target concentrations (figure 13) (30). Samples do not require post-amplification analysis and little prior knowledge of target abundance is required. Use a two-step RT-PCR strategy for quantifying multiple messages in a single RNA sample and use a one-step strategy for added convenience when processing large numbers of samples Cycle Number Panel B Total HeLa RNA 500 ng 50 ng 5 ng 500 pg Slope: Y-Intercept: Correlation Coefficient: pg 5 pg 0 Fluorescence Threshold Starting Quantity (pg total HeLa RNA) FIGURE 13. Real-time PCR quantitation of retinoblastoma mrna. RT-PCR of duplicate samples of total HeLa RNA was performed with the PLATINUM Quantitative RT-PCR THERMOSCRIPT One-Step System (cdna synthesis was performed at 60 C) and detected using a FAM-labeled Molecular Beacon probe (400 nm) on an ABI Prism Panel A. Relative fluorescence intensity of duplicate samples during PCR. Panel B. Standard curve. RT-PCR APPLICATIONS 9

10 C HAPTER 5 Improving PCR Specificity Primer Design Careful primer design is one of the most important aspects of PCR. The ideal primer pair anneals to unique sequences that flank the target and not to other sequences in the sample. Poorly designed primers may amplify other, nontarget sequences. The following guidelines describe the desirable characteristics of a primer sequence that increase specificity: Typical primers are 18 to 24 nucleotides in length. The primer needs to be long enough for the sequence to be unique and to reduce the probability of the sequence being found at non-target sites. However, primers greater than 24 nucleotides do not confer greater specificity. Longer sequences can hybridize with some mismatching, which decreases specificity, and hybridize slower than shorter sequences, which may decrease yield (2). Select primers that are 40% to 60% GC or mirror the GC content of the template. Design primers with G or C residues in the 5 and central regions. This increases the primer s stability and confers hybridization stability with the target sequence. Avoid complementary sequences at the 3 end of primer pairs. This prevents amplification from the primers themselves to form primer-dimers. Avoid a GC-rich 3 end. Design primers to contain 3 As or Ts within the last 5 nucleotides (35). Avoid mismatches at the 3 end. The last 3 nucleotide needs to anneal to the template for the polymerase to catalyze extension (36). Avoid sequences with the potential to form internal secondary structure. This destabilizes primer annealing. Additional sequences that are not present on the target, such as restriction sites and promoter sequences, can be added to the 5 end of a primer without affecting specificity. These sequences are not included when estimating the T m of a primer. However, check Careful primer design is one of the most important aspects of PCR. Poorly designed primers may amplify other, non-target sequences. these regions for complementarity and internal secondary structures. Sometimes only limited sequence information is available for primer design. For example, if only the amino acid sequence is known, degenerate primers are designed. Degenerate primers are a mix of different sequences representing all possible bases coding for a single amino acid. To improve specificity, minimize the degeneracy consulting codon usage tables for base preference in different organisms (2). In addition, deoxyinosine may be used in places where more than one base is possible. Inosine pairs with all bases and will lower the annealing temperature of the primer. Do not include degenerate bases at the 3 end of the primer, since annealing of the last three bases on the 3 end can be enough to initiate PCR at the wrong sites. Use higher primer concentrations (1 µm to 3 µm) because many of the primers in the degenerate mixture are not specific for the target (37). Primer Annealing Temperature Another important parameter for primers is the melting temperature (T m ). This is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The T m is necessary to establish an annealing temperature for PCR. Ideally, the annealing temperature is low enough to guarantee efficient annealing of the primer to the target, but high enough to minimize nonspecific binding. Reasonable annealing temperatures range from 55 C to 70 C. Annealing temperatures are generally set about 5 C below the T m of the primers. There are several formulas for estimating the T m (38-40). Table 4 lists two of the commonly used formulas for determining a primer s T m. The first formula was derived for hybridization in high salt (1 M) and is valid for primers <18 bases. The second formula estimates T m based on GC content and salt concentration. The most reliable method for determining the T m of a primer is the nearestneighbor analysis (38). This method predicts the hybridization stability of a primer from Table 4 Simple Formula (39) (valid for primers <18 bases) T m = 4 C (G + C) + 2 C (A + T) T m for Oligonucleotides (40) (dependent on salt concentration) T m = (log 10[Na + ]) (%G + C) 675/n Where n = number of bases and [Na + ] = monovalent (Na + or K + ) cations TABLE 4. Formulas to estimate Tm. the primary sequence and the identity of the neighboring bases. Most computer programs use nearest-neighbor analysis. The T m can vary significantly depending on the formula used and the primer sequence. Since most formulas provide an estimated T m value, the annealing temperature is only a starting point. Specificity can be increased by analyzing several reactions with increasingly higher annealing temperatures. Begin at 5 C below the estimated T m and increase the annealing temperature in 2 C increments. Higher annealing temperatures can reduce the formation of both primer-dimer and nonspecific products. For best results, both primers need to have a similar T m. Primer pairs whose T m varies by more than 5 C exhibit greater mispriming due to the 10 IMPROVING PCR SPECIFICITY

11 C HAPTER 5 use of lower annealing temperatures during cycling. If two primers have a different T m s, set the annealing temperature 5 C below the lowest T m. Or to increase specificity, perform 5 cycles at the annealing temperature established by the primer with the higher T m and then the remaining cycles at the annealing temperature established by lower T m. This allows some copies of the target to be generated under more stringent conditions. Touchdown PCR Touchdown PCR increases sensitivity by using stringent annealing conditions during the early cycles of PCR (41). Cycling begins with annealing temperature approximately 5 C above the estimated T m. It is incrementally decreased by 1 C to 2 C every cycle until the annealing temperature is about 5 C below the T m. Only targets with the greatest homology will be amplified. These products will continue to be amplified and will compete out nonspecific products amplified in later cycles. Touchdown PCR is useful in applications where the degree of homology between the target sequences and the primer are unknown, such as AFLP DNA fingerprinting. Primer Concentration Primer concentration can affect specificity. Optimal primer concentration typically falls between 0.1 to 0.5 µm. Higher concentrations of primer may result in the amplification of nonspecific products. To determine primer concentration, read the optical density at 260 nm (OD 260 ). Then, using Beers Law (formula 1) calculate the concentration by using the absorbance and the reciprocal of the millimolar extinction coefficient (nmol/od). The millimolar extinction coefficient can be calculated using formula 2 (42). Unlike large doublestranded DNA molecules where an averaged extinction coefficient can be used, the use of the extinction coefficient calculated for the primer is essential to accurately determine the concentration (42). This is because primers are short and the base composition can vary greatly. Both the extinction coefficient (OD/µmol) and the reciprocal of the extinction coefficient (µmol/od) are provided on the GIBCO BRL Custom Primer certificate of analysis. In addition, do not estimate primer concentration on ethidium bromide-stained gels using oligonucleotides as standards, since the staining capability of the standard and the primer can vary greatly depending on their sequence (43). Application PCR Table 5 First-strand cdna synthesis for RT-PCR AFLP technology PCR using primers with 5 sequences (restriction endonuclease sites, RNA polymerase promoter sites, etc) PCR primers >50 bases Cycle sequencing Isothermal sequencing Site-directed mutagenesis CFLP technology Formula 1 Concentration= A 260 dilution factor the reciprocal of the extinction coefficient conversion factors Example: To calculate the concentration of an oligonucleotide in 1 ml, measure the A 260 of 10 µl of the oligonucleotide in 990 µl of water (1:100 dilution). If the A 260 = 0.14 OD and the oligonucleotide has a reciprocal extinction coefficient of 4.9 nmol/od, the concentration would be calculated as follows: Concentration = 0.14 OD nmol 1 µmol 10 3 ml ml OD 10 3 nmol L = 69 µm Formula 2 Millimolar Extinction Coefficient of Oligonucleotide = A(15.2) + C(7.05) + G(12.01) + T(8.4) at ph 8.0 Where A, C, G, and T are the number of das, dcs, dgs, and dts (35). Minimum Suggested Purity TABLE 5. Minimum recommended primer purity for PCR applications. Primer Purity and Stability Standard purity Custom Primers are sufficient for most PCR applications (table 5). With Life Technologies PARALLEL ARRAY SYNTHESIS technology, desalting is not necessary. The benzoyl and isobutyryl groups removed by desalting are found in lower amounts with PARALLEL ARRAY SYNTHESIS technology, compared to other methods, and thus do not interfere with PCR. Some applications require purification to remove any less than full-length sequences generated during synthesis. These truncated sequences are generated because DNA synthesis chemistry is not 100% efficient. It is a cyclical process in which DNA is synthesized 3 5 using chemical reactions that are repeated for each base added. Failures can occur during any cycle. Longer primers, especially primers >50 bases, have a greater portion of truncated sequences and may require purification. Primer yield is affected by the efficiency of the synthesis chemistry and by the method of purification. Life Technologies guarantees the total oligonucleotide yield as a minimum number of OD units (table 6, page 12). Custom Primers are shipped lyophilized. It is best to reconstitute primers in TE [10 mm Tris-HCl (ph 8.0), 1 mm EDTA] to bring the final concentration to 100 µm. TE is better than deionized water since the ph of the water is often slightly acidic and can cause hydrolysis of the oligonucleotide. The stability of the primer depends on storage conditions. Store lyophilized and reconstituted primers at 20 C. Primers Standard Standard reconstituted in TE at 10 µm Standard are stable for >6 months at Cartridge 20 C, but only <1 week when PAGE stored at room temperature Standard (15 C to 30 C). Lyophilized Desalted primers are stable >1 year at Cartridge 20 C, and up to 2 months Desalted when stored at room temperature (15 C to 30 C). IMPROVING PCR SPECIFICITY 11

12 C HAPTER 5 Improving PCR Specificity (continued) Hot Start Hot-start PCR is one of the most important methods, in addition to good primer design, for increasing PCR specificity. Even though the optimal extension temperature for Taq DNA polymerase is approximately 72 C, the polymerase has activity at room temperature (44). Thus, nonspecific products are often generated during PCR set-up and at the start of thermal cycling when reactions are briefly incubated at temperatures well below the annealing temperature (45,46). Once these nonspecific products are formed they can be efficiently amplified. Hot-start PCR is particularly effective when the sites available for designing primers are limited due to the location of genetic elements, such as sitedirected mutagenesis, expression cloning, or the construction and manipulation of genetic elements for DNA engineering. A popular method that limits Taq DNA polymerase activity is to set-up amplification reactions on ice and place them into a preheated thermal cycler. This method is simple and inexpensive, but does not completely inactivate enzyme activity and therefore does not completely eliminate the amplification of nonspecific products. Hot start delays DNA synthesis by withholding one of the essential components until the thermal cycler reaches the denaturation temperature. Most manual hot-start methods involve the delayed addition of Taq DNA polymerase, which can be cumbersome especially for high-throughput applications (46). Other hot-start methods use wax barriers to encapsulate an essential component, such as magnesium or enzyme, or to physically separate reaction components, such as template and buffer, from each other. Melting of the wax during thermal cycling releases and mixes all of the components together. Like manual hot-start methods, wax barrier methods can be cumbersome, prone to contamination and unreliable in high-throughput applications. PLATINUM DNA Polymerases are convenient and highly-effective for automatic hot-start PCR (figure 14). PLATINUM Taq DNA Polymerase is comprised of recombinant Taq DNA polymerase complexed with monoclonal antibodies to Taq DNA polymerase (47). The antibodies prevent enzyme activity during PCR set-up and even during prolonged incubations at room temperature. 100 bp DNA Ladder Panel A kb 114 bp Panel B 1 Kb DNA Ladder FIGURE 14. Improved specificity with PLATINUM Taq DNA Polymerase. Panel A. Detection of cloned HIV DNA in human genomic DNA. 1,000 copies of plasmid DNA with the HIV gag region was mixed with 100 ng of genomic DNA and amplified with primers to the gag region. Lane 1. Taq DNA polymerase with room temperature assembly. Lane 2. Taq DNA polymerase with manual hot start by addition of enzyme at 94 C. Lane 3. PLATINUM Taq DNA Polymerase with room temperature assembly. Panel B. Amplification of 4.1 kb of human β-globin from 100 ng of human genomic DNA. Lane 1. Taq DNA polymerase with room temperature assembly. Lane 2.Taq DNA polymerase with assembly on ice and placed in a preheated (80 C) thermal cycler. Lane 3. PLATINUMTaq DNA Polymerase with room temperature assembly. Taq DNA polymerase is released into the reaction after incubation at 94 C during the denaturation step, restoring full polymerase activity. In comparison to chemically modified Taq DNA polymerase for hot-start PCR, PLATINUM enzymes do not require prolonged incubation at 94 C (10 to 15 min) to activate the polymerase. Greater than 90% of Taq DNA polymerase activity is restored in 2 min at 94 C with PLATINUM Taq DNA Polymerase (48). Table 6 Minimum Yield (OD) for Different Primer Purities* Standard/ Number of Bases Synthesis Scale Desalted Cartridge HPLC PAGE <20 50 nmole 2 2 NA NA 200 nmole µmole µmole 200 NA nmole 5 2 NA NA 200 nmole µmole µmole 500 NA Note: For primers >50 bases, PAGE purification is recommended and HPLC is not an option. NA is not available. *These yields are seen with the following 5 modifications: biotin, fluorescein (FITC), rhodamine, primary amines (NH 2), phosphate (PO 4), HEX, TET, FAM, and phosphorothioates (S-Oligos). Other modifications may have slightly lower yields. TABLE 6. Minimum oligonucleotide yield for the various purification methods. Magnesium Concentration Magnesium affects several aspects of PCR including DNA polymerase activity, which can affect yield; and primer annealing, which can affect specificity. The dntps and template bind magnesium and reduce the amount of free magnesium available for enzyme activity. The optimal magnesium concentration varies for each primer pair and template, but the starting concentration in typical PCR containing 200 µm dntps is 1.5 mm (Note: for real-time quantitative PCR use 3 to 5 mm magnesium with fluorescent probes) (2). Higher concentrations of free magnesium can result in greater yield, but can also increase nonspecific amplification and reduce fidelity (49,50). To determine the best concentration, perform a magnesium titration using from 1 mm to 3 mm in 0.5 mm increments. To reduce the 12 IMPROVING PCR SPECIFICITY

13 C HAPTER kb need for magnesium optimization, use PLATINUM Taq DNA Polymerase. PLATINUM Taq DNA Polymerase functions over a broader range of magnesium concentration than Taq DNA polymerase so less optimization is required (figure 15) (47). Additives to Enhance PCR Optimization of the annealing temperature, primer design, and magnesium concentrations is adequate to achieve high specificity amplification of many targets. However, some targets, including those with a high- GC content, require additional measures. Additives that affect DNA melting temperature provide another method for improving product specificity and yield (figure 16). Complete denaturation of the template is Panel A (mm MgCl2) Panel B required to obtain the best results. Additionally, secondary structure can prevent primer binding and enzymatic elongation. PCR additives, including formamide, DMSO, glycerol, betaine, and PCR X Enhancer Solution can enhance amplification (51-53). Their proposed mechanism is to lower the melting temperature, thereby aiding primer annealing and helping the DNA polymerase extend through regions of secondary structure (53). PCR X Solution has additional benefits. It requires less magnesium optimization and works with PLATINUM Taq DNA Polymerase (figure 16) and PLATINUM Pfx DNA Polymerase. Thus, combining PLATINUM technology and additives enhances specificity while minimizing the need for the third approach-magnesium optimization. For best results, optimize the concentration of the additives (figure 17), especially DMSO, formamide, and glycerol that inhibit Taq DNA polymerase (46,52). Nested PCR Sequential rounds of amplification using nested primers can improve specificity and sensitivity (3). The first round is a standard amplification of 15 to 20 cycles. A small aliquot of the initial amplification is diluted 1:100 to 1:1,000 and added to the second (mm MgCl2) FIGURE 15. Broader magnesium range with PLATINUM Taq DNA Polymerase. A 2.8-kb region of the human β-globin gene was amplified from 100 ng of human genomic DNA. Panel A.Taq DNA polymerase with assembly on ice and placed in a preheated (80 C) thermal cycler. Panel B. PLATINUM Taq DNA Polymerase with room temperature assembly. round of amplification with 15 to 20 cycles. Alternatively, the initial amplification product can be size selected by gel purification. Two different primer sets are used for the two rounds of amplification. The second amplification uses a nested set of primers that bind to the target just inside the first set of primers. The chance of amplifying multiple targets is reduced with nested PCR since fewer targets will be complementary to both sets of primers; whereas performing the same total number of cycles (30 to 40) with the same set of primers often amplifies nonspecific targets. Nested PCR can increase the sensitivity from limited amounts of target, such as amplifying a rare message, and increase the specificity of more challenging PCR applications such as 5 RACE. Panel A Panel B 156 bp 62% GC FIGURE 16. Improved specificity with PCR x Enhancer Solution. A 156-bp fragment (62% GC content), was amplified from 100 ng of human genomic DNA using PLATINUM Taq DNA Polymerase. MgCl 2 was titrated (1.0, 1.5, 2.0, 2.5 mm, lanes 1 to 4 respectively) using: standard PCR buffer (panel A) or PCR x Amplification Buffer with 1X PCR x Enhancer Solution (panel B). Cycling was 35 cycles of 94 C for 30 s; 60 C for 30 s, and 68 C for 1 min. 100 bp DNA Ladder 0X 1X 2X 2.5X 3X 3.5X 4X 149 bp 78% GC FIGURE 17. Titration of PCR x Enhancer Solution. A 149-bp (78% GC) trinucleotide repeat-containing sequence was amplified from 100 ng of human genomic DNA with PLATINUM Taq DNA Polymerase in 1X PCR x Amplification Buffer with varying amounts of PCR x Enhancer Solution (0 to 4X). IMPROVING PCR SPECIFICITY 13

14 C HAPTER 6 Increasing PCR Sensitivity Template Quality Template quality affects product yield. A number of contaminants found in DNA preparations can inhibit PCR (46). Reagents such as SDS, which are used in standard genomic DNA preparations, can inhibit amplification reactions at concentrations as low as 0.01%. Newer methods for isolation of high-quality genomic DNA include DNAZOL Reagents (53), which are guanidine-detergent lysing solutions, and the FTA Products, which is a matrix-bound method for the storage and purification of DNA from blood and other biological samples (table 7) (55). Template Concentration The amount of starting template is important for obtaining good product yields. For most amplifications, 10 4 to 10 6 starting target molecules allows sufficient amplification to visualize the product on an ethidium bromide-stained gel (2). The optimal amount of template required depends on the size of the genome (table 8) (56). For example, 100 ng to 1 µg of human genomic DNA, correlating to to molecules, is sufficient to detect a PCR product from a single-copy gene. For plasmid DNA, which is much smaller, the amount of DNA added to PCR is in the picogram range. Enzyme Choice In addition to using high-quality template DNA, the choice of polymerase also affects yield (see table on page 22). PLATINUM polymerases provide better yield than other polymerases because they prevent amplification of nonspecific product during PCR set-up (figure 18). For high-sensitivity PCR of long products (up to 12 kb), choose an enzyme mix, preferably in a PLATINUM format, such as PLATINUM Taq DNA Polymerase High Fidelity. This enzyme combines the benefits of PLATINUM technology with those of enzyme mixes (Taq DNA Polymerase mixed with a proofreading polymerase). 1 Kb PLUS DNA LADDER Taq DNA Polymerase PLATINUMTaq DNA Polymerase PLATINUMTaq DNA Polymerase High Fidelity FIGURE 18. Amplification with different thermostable polymerases. Human blood was spotted on FTA Cards. DNA was amplified directly from 1 to 3 mm punches of washed FTA Cards in 50 µl using Taq DNA polymerase (panel A), PLATINUM Taq DNA Polymerase (panel B), and PLATINUM Taq DNA Polymerase High Fidelity (panel C). Amplified products were 4.1, 5.2, 7.5, 8.0, and 8.4 kb in lanes 1 to 5, respectively. Method Proteinase K/phenol extraction DNAZOL Reagents FTA Products TABLE 7. Methods for genomic DNA isolation. Table 8 Target Molecules/µg Amount of DNA (µg) Genomic DNA Size (bp)* of Genomic DNA for ~10 5 Molecules E. coli Saccharomyces cerevisiae Arabidopsis thaliana Drosophila melanogaster Homo sapiens Xenopus laevis Mus musculus Zea mays puc 18 plasmid DNA * Haploid genome size TABLE 8. Correlation of genome size and number of molecules. Table 7 kb Description Classic method Fast, phenol-free DNA isolation Matrix-bound purification and storage 14 INCREASING PCR SENSITIVITY

15 C HAPTER 7 Improving Fidelity Enzymes with Proofreading PCR applications involving cloning and sequence analysis, expression of PCR products, or site-directed mutagenesis require high-fidelity PCR. Taq DNA polymerase is considered a lowfidelity polymerase since it lacks 3 to 5 exonuclease (proofreading) activity. The use of thermostable polymerases with 3 exonuclease activity improve fidelity (57,58). However, these polymerases can give lower yields than Taq DNA polymerase. PLATINUM Pfx DNA Polymerase has significantly better fidelity than Taq DNA polymerase and offers the advantage of high yield and specificity of PLATINUM products (figure 19) (59). 1 Kb PLUS DNA LADDER 1 Kb PLUS DNA LADDER Panel A Panel B Panel C FIGURE 19. High sensitivity and specificity with PLATINUM Pfx DNA Polymerase. Genomic DNA (100, 50, 10, 5, 1, and 0 ng, lanes 1 to 6) was amplified with primers for a 1.1-kb fragment of human thrombospondin for 35 cycles. Panel A. 1 unit of PLATINUM Pfx DNA Polymerase with room temperature set-up. Panel B. 2.5 units of PfuTurbo DNA Polymerase with set-up on ice. Panel C. 1 unit of Taq DNA Polymerase with set-up on ice. 1 Kb PLUS DNA LADDER 1.1 kb Enzyme Mixes Mixing Taq DNA polymerase with a second polymerase with 3 exonuclease activity provides greater fidelity than Taq DNA polymerase alone and allows for higher yield and amplification of longer templates. The GIBCO BRL high fidelity enzyme mix, PLATINUM Taq DNA Polymerase High Fidelity has 6-times greater fidelity than Taq DNA polymerase alone and can amplify up to 12 kb. Other Parameters Besides enzyme, high dntp or magnesium concentrations can reduce fidelity. Decreasing the concentration of dntps from 200 µm to µm can increase accuracy. If the concentration is not the same for all four nucleotides, the fidelity will be effected. Performing fewer cycles of PCR also can help increase fidelity, since the probability of a mutation increases with increasing cycle number and product length. IMPROVING FIDELITY 15

16 C HAPTER 8 Other Things to Consider Amplifying Long Targets When amplifying targets >5 kb, use an enzyme or enzyme mix for long PCR to obtain the best yield (see table on page 22 and figure 18 on page 14). Taq DNA polymerase does not efficiently amplify longer targets (>5 kb), presumably because it lacks 3 5 exonuclease activity and cannot correct dntp misincorporations (60). The elongation rate from a mismatch is greatly reduced, which decreases the yield of longer products. Mixing Taq DNA polymerase with a thermostable DNA polymerase containing 3 5 exonuclease activity can allow The Concert Rapid PCR Purification System purifies PCR fragments from 80 bp to 10 kb. amplification of targets up to 30 kb (61). PLATINUM Pfx DNA Polymerase (has 3 5 exonuclease activity) can also amplify longer products ( 12 kb). In addition to choosing the correct thermostable polymerase, the amplification of longer products requires changes to the extension times, denaturation times, and buffer ph of the standard protocol. Increase extension times to 1 min/kb to allow the polymerase to complete synthesis. Typically the extension temperature is lowered to 68 C for more effective long PCR (60,61). Since the elongation times are long, up to 20 min for a 20-kb target, a buffer with a higher initial ph is used. If the ph falls below ph 7.0, the DNA can depurinate. To protect the template against damage, minimize the denaturation time at 94 C to 30 s or less during each cycle and limit the preamplification denaturation time to 94 C for 1 min. Primers are designed in the same manner as those used in a standard protocol, with primers 18 to 24 bases giving good product yields (60,61). Prevention of Carry-Over Contamination PCR is susceptible to contamination problems because it is a sensitive amplification technique. Small amounts of contaminating DNA from an exogenous source can be amplified along with the desired template. A common source of contamination occurs when previously amplified products are introduced into new amplification reactions. This is called carry-over contamination. Purified DNA from other samples and cloned DNA can also be sources of contamination. Carry-over contamination can be minimized by using good laboratory procedures during PCR. Use aerosol-resistant tips to prevent aerosols from reaching the pipette barrel. Designate separate areas for PCR sample set-up and post-amplification analysis and change gloves before preparing new reactions. Always perform a negative control, without template, to check for contamination. Use premixed reaction components instead of adding each reagent to individual reactions. One method to prevent carry-over contamination uses uracil DNA glycosylase (UDG) (62). This enzyme (also known as uracil-n-glycosylase or UNG) removes uracil residues from DNA. Substituting deoxyuracil for thymine in amplification reactions allows previously amplified products to be distinguished from template DNA. Since the previously amplified products are susceptible to UDG, newly assembled reactions are treated with UDG before PCR to destroy carry-over products. Purification of PCR Products Residual reaction components including primers, nucleotides, and thermostable polymerases can inhibit cloning, sequencing, and labeling reactions. Thus, it is often necessary to purify PCR products before further manipulation. Clean-up processes involving multiple phenol:chloroform extractions followed by PEG precipitation (63) or isopropanol precipitation (2) are effective but time consuming and can result in product losses. The CONCERT Rapid PCR Purification System purifies PCR products from reaction components in 10 min. It is effective for a wide range of PCR fragments, from 80 bp to 10 kb, and provides efficient recovery of up to 95% of the original sample (figure 20) (64). Some PCR products may require purification, from nonspecific PCR products that can interfere with cloning or sequencing, by gel electrophoresis. The desired product is purified from the agarose using the CONCERT Gel Extraction System, a silicabased technology for rapid isolation of DNA fragments from gels. When cloning PCR products by restriction endonucleases, TAQUENCH PCR Cloning Enhancer provides an alternative to the clean-up of PCR products. If 3 -recessed termini are generated by digestion, residual Taq DNA polymerase and dntps from the PCR can fill in the 3 -recessed termini (65). This lowers the cloning efficiency and generates clones ligated in the improper reading frame. However, the addition of TAQUENCH Enhancer before restriction digestion minimizes Taq DNA polymerase activity for efficient cloning (65). High DNA MASS Ladder 1 2 A B A B kb primers FIGURE 20. Purification of PCR fragments following amplification. Completed PCRs containing ~1 µg of amplified product were spiked with 1.2 µg of primer (lane 1) or 3.5 µg of primer (lane2). Primers were 36 to 40 bases. Spiked reactions were purified, and aliquots of the eluate before (lane A) and after (lane B) purification were analyzed by agarose electrophoresis. 16 OTHER THINGS TO CONSIDER

17 C HAPTER 9 PCR Applications Multiplex PCR In multiplex PCR, multiple primer sets are used simultaneously to amplify several different loci. This complex PCR often results in low product yield and requires higher concentrations of magnesium (67). A false negative result may be obtained primer (nm) template (ng) M bp Panel A FIGURE 21. PLATINUM Taq DNA Polymerase provides greater sensitivity for multiplex PCR. Multiplex amplification of the dystrophin gene was performed with 5 different primer sets from human genomic DNA withtaq DNA polymerase (panel A) or PLATINUM Taq DNA Polymerase (panel B). Using the indicated primer and template concentration, products ranging from 268 bp to 547 bp were amplified from human genomic DNA. if reactions are not optimized correctly. The increased robustness of PLATINUM Taq DNA Polymerase over a broad range of magnesium concentrations increases success in multiplex reactions (figure 21). Genotyping with Dinucleotide Repeat Markers Determining the size of the amplified dinucleotide repeat region can be challenging since Taq DNA polymerase often adds a nontemplated nucleotide to PCR products. The fraction of products in an amplification reaction containing an extra nucleotide is sequence and primer dependent and can vary greatly. However, PLATINUM GENOTYPE Tsp DNA Polymerase exhibits minimal nontemplated nucleotide addition to help increase the accuracy of allele size determination (figure 22) (66). Simply substitute PLATINUM GENOTYPE Tsp DNA Polymerase for Taq DNA polymerase in these amplifications. M Panel B Tools for Detecting Polymorphisms Amplified Restriction Fragment Polymorphism (AFLP) technology is a technique for fingerprinting genomic DNA from plants and microorganisms (figure 23) (68,69). RFLP, AP-PCR, and RAPD are also common DNA fingerprinting techniques. RFLP is a hybridization-based method which is time consuming and requires a large amount of genomic DNA. AP-PCR and RAPD are faster, PCR-based methods, but both are sensitive to reaction conditions which can affect the reproducibility of the techniques. AFLP technology combines the reliability of RFLP and the convenience of a PCR-based fingerprinting method for a more robust and informative method. DNA fingerprints generated with AFLP technology can be used as a tool to identify a specific DNA sample or to assess the similarity between samples (figure 24) (69). A number of parameters influence AFLP results, including the number of selective nucleotides in the primers (69). The number of amplified bands decreases when the number of selective nucleotides increases. Larger genomes require more selective nucleotides to amplify an appropriate number of DNAs, since more restriction fragments are generated from digesting a larger genome. The template quality can also affect AFLP results. Poor quality template may inhibit restriction endonucleases, resulting in incomplete digestion and fewer amplified bands visible on the gel (69). However, the AFLP technique is tolerant of variation in template concentration, with no difference observed with amounts of DNA between 100 ng to 5 µg. Fluorescence Intensity 2,500 2,000 1,500 1, ,500 2,000 1,500 1, Figure 21 Nucleotides Taq DNA Polymerase n+1 (66% n + 1) n PLATINUM GENOTYPE Tsp DNA Polymerase (0% n + 1) FIGURE 22. Comparison of DNA polymerases in genotyping of dinucleotide repeat markers. Amplification of nga106 locus of Arabidopsis thaliana generated withtaq DNA polymerase (66% n + 1) or PLATINUM GENOTYPE Tsp DNA Polymerase (0% n + 1). 5 3 GAATTC CTTAAG AATTC G TTAA EcoR I adapter primer +1 5 A AATTCN TTAAGN preselective amplification with EcoR I primer +A Mse I primer +C primer +3 5 AAC AATTCA TTAAGT selective amplification with primers +3 AATTCAAC TTAAGTTG Figure 23 n n+1 TTAA AATT +EcoR I Mse I T AAT +EcoR I adapter Mse I adapter TA Mse I adapter NTTA NAAT C 5 GTTA CAAT AAC 5 TTGTTA AACAAT denaturing polyacrylamide gel electrophoresis Mse I adapter sequences 3 5 EcoR I adapter sequences FIGURE 23. Schematic of the AFLP Analysis System FIGURE 24. A typical AFLP fingerprint. The polymorphism of soybean ecotypes was performed using the AFLP System I and the selective primers EcoR I+ACC and Mse I+CAA. Ecotypes were Holladay; Morgan; Hytest; Essex; Bass; T135; P88; PI88788; P83; PI83495; P90; PI90763; lanes 1 to 9, respectively. PCR APPLICATIONS 17

18 C HAPTER 10 Troubleshooting Guide Problem Possible Cause Suggested Solution RT-PCR Sensitivity: Little or no RT-PCR product visible after agarose gel analysis. PCR Sensitivity: Little or no PCR product visible after agarose gel analysis. RNA was degraded RNA contained an inhibitor of RT Polysaccharide coprecipitation of RNA Primer used for first-strand cdna synthesis did not anneal well Not enough starting RNA RNA template had high secondary structure The primers or template are sensitive to remaining RNA template Target not expressed in tissue analyzed PCR did not work Poor PCR primer design DNA contains inhibitors GC-rich template Template concentration is too low Magnesium concentration is too low Analyze RNA on a denaturing gel before use to verify integrity. Use good aseptic technique for RNA isolation. Process tissue immediately after removal from animal. Store RNA in 100% formamide (15). If using placental RNase inhibitor, do not heat >45 C or use >ph 8.0 or inhibitor may release any bound RNases. Also, DTT must be present when the RNase inhibitor is added at 0.8 mm DTT. Remove inhibitor by ethanol precipitation of the RNA. Include a 70% (v/v) ethanol wash of the RNA pellet. Glycogen (0.25 µg to 0.4 µg/µl) can be included to aid in RNA recovery for small samples. Inhibitors of RT include: SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, and guanidinium salts (9). Test for inhibitors by mixing a control RNA with the sample and comparing yields to control RNA reaction. Precipitate RNA with lithium chloride to remove polysaccharides. Be sure annealing temperature is appropriate for your primer. For random hexamers, a 10 min incubation at 25 C is recommended before incubating at reaction temperature. For gene-specific primers (GSP), try another GSP or switch to oligo(dt) or random hexamers. Make sure GSP is the antisense sequence. Increase the amount of RNA. For <50 ng RNA, use 0.1 µg to 0.5 µg acetylated BSA in first-strand cdna synthesis (21). Denature/anneal RNA and primers in the absence of salts and buffer. Raise the RT reaction temperature up to 50 C for SUPERSCRIPT II RT or up to 65 C for THERMOSCRIPT RT. Note: Do not use oligo(dt) as a primer over 60 C and choose a GSP that will anneal at your reaction temperature. For RT-PCR products >1 kb, keep reaction temperature 65 C. Note: Do not use M-MLV RT above 37 C. Use random hexamers in the first-strand reaction if full-length cdna is not needed. RNase H treat first-strand cdna before PCR (14). Try a different target or tissue. For two-step RT-PCR, do not use more than 1 /5 of the RT reaction in the PCR step. Avoid complementary sequences at the 3 end of primers. Avoid sequences that can form internal hairpin structures. Design primers with similar T ms. Reagents such as DMSO, SDS, and formamide can inhibit Taq DNA polymerase. If inhibitor contamination is suspected, ethanol precipitate the DNA sample. For templates >50% GC content, use PCR x Enhancer Solution. Start with 10 4 copies of the target sequence to obtain a signal in 25 to 30 cycles. Determine the optimal magnesium concentration for each template and primer pair by performing a reaction series from 1 mm to 3 mm in 0.5 mm increments. Note: Use 3 mm to 5 mm magnesium for real-time quantitative PCR. 18 TROUBLESHOOTING GUIDE

19 C HAPTER 10 Problem Possible Cause Suggested Solution PCR Sensitivity: Little or no PCR product visible after agarose gel analysis. (continued) RT-PCR Specificity: Unexpected bands after gel analysis PCR Specificity: Unexpected bands after agarose gel analysis PCR Fidelity: PCR induced errors found in the product sequence Annealing temperature is too high Primer concentration is too low Nonspecific annealing of primers to templates Poor GSP design Genomic DNA contamination of RNA Primer-dimer formation Nonspecific annealing of primers to template Magnesium concentration is too high Primer mispriming due to amplification from complex templates. Contaminating DNA from an exogenous source Primer binding sites are inaccessible due to secondary structure Polymerase has low fidelity Too many cycles The concentration of all four deoxynucleotides is not equal Use the equations listed in table 4 (page 10) to estimate the T m and set the annealing temperature 5 C below the T m. Since these equations estimate T m values, the true annealing temperature may actually be higher or lower. Optimal primer concentration is between 0.1 µm to 0.5 µm. To accurately determine primer concentration, read the optical density at 260 nm (OD 260). Then, calculate the concentration using the absorbance and the extinction coefficient (see page 11). Use a GSP instead of random hexamers or oligo(dt) for first-strand synthesis. Try a GSP that allows cdna synthesis at high temperatures. Follow the same rules as described for amplification primers. Treat RNA with DNase I, Amplification Grade (see page 7). Check for DNA contamination with a control reaction without RT. Design primers without complementary sequences at the 3 ends. Increase the annealing temperature in 2 C to 5 C increments and minimize the annealing time. Use higher annealing temperatures for the first few cycles, followed by lower annealing temperatures. Use PLATINUM Taq DNA Polymerase for automatic hot-start PCR (47). Avoid 2 or 3 dgs or dcs at the 3 end of primers. Optimize magnesium concentration for each template and primer combination. Use nested primers or touchdown PCR. Use aerosol-resistant tips and UDG (see page 16). For templates >50% GC content use (1X-3X) PCR X Enhancer Solution. Use a proofreading thermostable polymerase such as PLATINUM Pfx DNA Polymerase. Reduce cycle number. Prepare a new deoxynucleotide mix and ensure that the concentration of all four nucleotides is equal or use a prepared mix. TROUBLESHOOTING GUIDE 19

20 C HAPTER 11 References 1. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N. (1985) Science 230, Innis, M.A., Gelfand, D.H., Sinsky, J.J., and White, T.J. (1990) PCR Protocols, A Guide to Methods and Applications. Academic Press, San Diego, California. 3. Dieffenbach, C.W. and Dveksler, G.S. (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 4. Siebert, P.D. and Larrick, J.W. (1991) Nature 359, Liang, P. and Pardee, A.B. (1992) Science 257, Frohman, M.A. (1993) Methods Enzymol. 218, Foley, K.P., Leonard, M.W., and Engel, J.D. (1993) Trends in Genetics 9, Mocharla, H., Mocharla, R., and Hodes, M.E. (1990) Gene 93, Gerard, G.F. (1995) FOCUS 16, Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, Chomczynski, P. (1993) BioTechniques 15, Simms, D., Cizdziel, P.E., and Chomczynski, P. (1994) FOCUS 15, Bracete, A.M. and Fox, D.K. (1999) FOCUS 21, Fox, D.K. and Nathan, M. 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21 C HAPTER 11 Locating FOCUS References Many of the References listed on the previous page are FOCUS articles. These articles contain many details related to protocols to obtain data presented in this Guide. Locating Previous FOCUS Articles FOCUS Volumes 16 Current Issue can be found on our web site at Several of the Classic FOCUS articles (most requested by you through Technical Services) can also be found on the web. Other articles, can be obtained from Literature Fulfillment through your local Life Technologies Office. Viewing a FOCUS Article from our Web Site Finding the Article: When you click on the FOCUS journal on the Home Page, the table of contents for the most recent issue will appear with old issues in the left box. (Older issues not displayed there are found by clicking Archives). There is also a search box in the upper left which will allow you to do a full text search of FOCUS articles. Viewing the Article: To view the article, you will need the latest Adobe Acrobat Reader. If you do not currently have the Acrobat Reader, you can download it for free at: There is a link to this site in the paragraph at the top of the FOCUS table of contents. Follow the procedure to download the Acrobat Reader. There are 2 stages to the process: 1. Follow Adobe s directions for downloading the proper file to your local drive. (Note file location when saving.) 2. Locate file you saved and install the reader on your computer. Note: To install the reader, just double click on the file on your local drive and follow the prompts. The reader is accessible to you for future use. You will not need to download the reader again, unless you remove the program from your computer. Now you can locate the FOCUS article, click on it, and it will open on your screen. Cumulative FOCUS Subject Index There are 2 ways to find articles on a specific technique: 1. The web site has a built-in subject index for the articles on the web. Just use the Search box at the top of the FOCUS section of the web. 2. A Cumulative Subject Index is available for volumes You can find this document on our web site or contact Literature Fulfillment through your local Life Technologies Office. Instructions to Authors Looking for information on submitting articles to FOCUS? Check out the Instructions to Authors file in the FOCUS section of our web site. LOCATING FOCUS REFERENCES 21