Chap 4 Synthesis, Sequencing and Amplification of DNA

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1 Chap 4 Synthesis, Sequencing and Amplification of DNA Why chemical synthesis of DNA? [1] 1. Used as a probe for DNA detection (Hybridization and Southern blotting) 2. Synthesized DNA for the assembly of genes or gene fragment (if the gene sequence is difficult to clone) 3. Introduce mutation (in vitro mutagenesis) 4. Single-stranded oligonucleotides ( mer) as primers for PCR and DNA sequencing 5. ds sequences as linkers (two palindromic ss DNA that base pair to each other to form a restriction site to help cloning of DNA fragments). 6. Adapter sequence: allows cdna to be cloned by blunt-end ligation and later excised from the vector by a different RE allow the DNA to be transferred to and from vectors. 1

2 Chemical Synthesis of oligonucleotides (by phosphoramidite method) Has been automated by commercial DNA synthesizers (can be finished in 10 hr) Oligonucleotides of specific sequence can also be ordered from suppliers at low cost Skip the details I. Synthesis of genes For short genes (60-80 bp) Synthesize two complementary strands and anneal (the process of heating (denature) and slow cooling (renature)) dsdna. For larger genes (>300 bp) The efficiency of correct synthesis of long oligo nt. is low. Synthesize a set of partially overlapping, complementary oligonucleotides (20-60 nt long). The sequences of the oligonucleotides are designed to partially overlap, so they can anneal as a ds array after annealing. 2

3 II. DNA sequencing To determine the DNA sequence Can be used to compare the similarity of genes (conserved regions), or to ensure accurate cloning (or synthesis of genes). Essential in molecular cloning because DNA sequence is needed to devise strategies Sanger Method [2]: an enzymatic procedure, currently the method of choice ddntp: dideoxynucleoside triphosphate, man-made molecule, blocks continued synthesis of DNA chain because 3 -OH group is replaced, phosphodiester bonds won t form with the next incoming dntp. Note: dntp and NTP are for DNA and RNA synthesis (in vitro transcription). 3

4 Read from bottom to top Synthesize a primer (17-24mer) to a predetermined segment of a cloning vector near the cloned DNA, the primer is annealed to the cloned DNA. The reactions are carried out in 4 separate tubes each with different ddntp. In the tube w/ ddatp whose concen. is carefully controlled so ddntp is incorporated at every possible site. The reaction generates a set of DNA strands with different lengths, each ends with a specific ddntp. Formamide is added to stop the synthesis and prevent DNA strands from base pairing (denaturation is crucial otherwise mobility in the gel is changed) Polyacrylamide gel electrophoresis autoradiography 3 5 4

5 Note: The polyacrylamide gel is run in the presence of urea to prevent renaturation, gel is usually thin and long. Because the terminated DNA chains of different sizes are 32 P labeled, the gel is exposed to an X-ray film, only radiolabelled DNA fragments are shown. The label can be on the primer or on the dntp. If the radiolabel is on the dntp, the radioactivity is labeled throughout their lengths, rather than at one end more radioactive less DNA is needed for sequencing. Usually the primer sequence is positioned about nt away from the insertion site of the cloned DNA so one can recognize the known sequence at the start. The accuracy can be improved by sequencing both strands of the DNA. The bottom means the shortest strand, i.e. the base near the start point. Capillary Electrophoresis (CE) and DNA Analyzer: CE uses long capillary tubes to replace the slab ( 厚板 ) gels CE has more efficient heat dissipation than slab gels higher run voltage, faster run time and higher sample throughput (new machines can run 96 tubes at one time) Each terminator is labeled with a different dye, during the electrophoresis in the single capillary tube (four terminators are included in one tube), the fluorescent dyes are excited and the fluorescence is detected, the data is processed and analyzed. Automation (invented by Leory Hood of Caltech): automated polymer gel filler, sample injection, detection and analysis elimination of manual operations and increased run for higher consistency and reliability (Craig Venter uses this technique to revolutionize the DNA sequencing). Higher sensitivity less DNA per sample 5

6 Application: Human Genome Project (HGP) made possible by the automation of the sequencing method. In the HGP, up to 32 million fragments, each bp, were sequenced. Next generation sequencing III. PCR (Polymerase Chain Reaction) [3] Invented by Karry Mullis in the mid-80s Revolution enable to amplify the DNA (genes) to abundant quantity for analysis (no need to clone and amplify by cell culture). Requirements: 1. Template 2. 4 dntp 3. Two synthetic primers (10-30 nt) that are complementary to the regions on opposite strands that flank the target sequence the two primers specify the regions to amplify 4. Thermostable DNA pol that can withstand heating to 95 C (e.g. Taq DNA pol from bacterium Thermus aquaticus inhabiting in water at 75 C) Three major steps: denaturation, annealing, extension 6

7 Normally cycles are used amplify 2 30 ( 1 billion fold) the short desired fragment is almost 100% of the entire population (in PCR, the percentages of original and long templates will drop as the process progresses, so the end product is the DNA fragment specified by the primers). After n cycles 2 n-2 ( fold for n=30) desired DNA fragments (1 st and 2 nd cycle don t generate the desired fragments), but in reality, usually copies are obtained Low efficiency is due to Mismatch of primers (to generate wrong, shorter strand insufficient dntp for correct synthesis) Repeated thermocycling reduced enzyme activity 7

8 There are chances of misincorporation. Taq does not have the proofreading activity, so variants such as Pfu or PowerTaq that possess proofreading activity can be used. Primer design should have a minimum 2 structure that prevent annealing Thermocycler is now common to provide precise control of experiment conditions which may vary case by case. The original template can be obtained from purified DNA or crude cell lysate, as long as the primers are specific enough. IV. Applications of PCR [2] 1. Amplifying cloned DNA from vectors To check the accuracy of cloning and the amplified DNA can be introduced to other vectors. PCR is run to amplify the cloned DNA using primers flanking the cloned insert. The cloned DNA insert is amplified and may have different lengths 8

9 The products can be further checked by DNA sequencing or a second set PCR using one GSP and one flanking primer to verify. 2. Introducing unique restriction enzyme sites [1] 9

10 3. Creating a recombinant DNA molecule by sequential PCR amplifications [1] 10

11 4. Gene Cloning [2] 11

12 5. Identification of an organism associated with a disease (e.g. enterovirus) Design the primer based on the known sequence of the target DNA (e.g. a DNA sequence of the virus). 12

13 Run PCR using the crude samples (requires only a small quantity) and the primers. After PCR, a DNA fragment of a specific size that is equivalent to the lengths of target DNA will be amplified only if the target DNA (i.e. the virus) is present in the sample. Real-time PCR can be used to quantify the amount of the organisms. Ex: to detect flu, we should know a certain conserved (or variant) region and design primers. After PCR, we compare the lengths of the fragment with the lengths of the known sequences rapid (conventional methods by growing organisms in culture or by using MAb often take longer). The template DNA (or RNA) for PCR is often genomic DNA (or RNA) extracted from the cells or viruses and don t need further purification. Others: Detecting mutation Monitor cancer therapy Sex determination Many others. V. Directed Mutagenesis and Protein Engineering [3] Sometimes the expressed proteins are not well suited for a specific application due to the restrictions in physical and chemical properties. Obtain the protein (gene) from an organism that grows in an unusual environment. e.g. if α-amylase is used at high T (in the industrial production of sugar), we may isolate the gene from a bacterium that grows naturally at 90 C. the protein expressed from the new gene is stable at high T. Alternative: by mutagenesis and selection to specifically change the a.a. encoded, hoping to improve the properties of the proteins such as: 1. Michaelis const. (Km, reflects the tightness of substrate binding), and the max rate of conversion (V max ) under defined conditions. (Enzyme kinetics: V=V max [S]/(K m +[S]), where [S] is substrate concentration) 2. Thermal tolerance or ph stability of a protein enable the protein to be used under special conditions 3. Requirement for a cofactor for certain continuous industrial production process. 4. Resistance to cellular proteases simplify purification, recovery. 13

14 Very difficult to create a new protein, while it s feasible to modify the existing properties of a known protein. May need to change 2 or more a.a. which are far apart in the linear sequence but are in proximity as a result of protein folding. 3-D structures are important for prediction. Bioinformatics can help predict on the basis of deduced a.a. sequence simplifying the task of producing a protein. To date, directed mutagenesis is a trial and error process. Typically a library of proteins is generated and screened for the desired change. Site-Directed Mutagenesis (Oligonucleotide-Directed Mutagenesis) Bacteriophage M13: has a ss DNA genome, when M13 infects the cells, ss DNA is replicated to ds form (semi-conservative), but at the late stage, only the outer strand (+ strand) is synthesized. These + strands are packaged into particles and are easy to separate from the ds form. 1. Clone the gene in a bacteriophage M13 vector (ds form) transform virus replication the ss form (M13 +strand) is isolated from the phage particle and mixed w/ a synthetic oligo. 14

15 2. The oligo is complementary to a segment of the cloned gene, except for one nt, which specifies the point mutation. e.g. ATT (AUU in mrna) encodes Ile, is to be changed to CTT (encodes Leu codon CUU). The oligo nt can hybridize to the M13+ strand The 3 end of the oligo acts as a primer that uses the + strand as the template, DNA synthesis catalyzed by the Klenow fragment of E. coli pol I. 4. T4 DNA ligase joins the last nt added w/ the 5 end of the primer. 5. ds M13 DNA are transformed into E. coli, producing M13 particles and lysing cells producing plaques. replication is semi-conservative 1/2 of the phages carry the mutated gene identified by DNA hybridization (using the oligo nt as probe) or DNA sequencing 6. the ds form of M13 is isolated (by sucrose gradient ultracentrifugation) cut the mutated gene insert into E. coli plasmid for expression Problem: In reality, only 1~5 % of progeny plaques contain the mutated gene. 1 The hybridization can be facilitated if (1) oligo nt is in excess of M13 DNA; (2) mismatch is in the middle of the nt; (3) mixing at low T and high salt concentration. 15

16 Modification: Introduce into an E. coli strain defective in 2 enzymes. (1) dutpase (dut) [dutp] inside the cells a few dutp incorporated into DNA in lieu of dttp (2) uracil N-glycosylase (ung) the incorporated dutp cannot be removed without ung In vitro w/o dutp, the 2nd strand has no U incorporated. The ds DNA is introduced into wt E. coli that has ung the original + strand is degraded only the mutated form is not degraded and continues to be replicated. enrich the yield of M13 carrying a sitespecific mutation. PCR-Amplified Oligonucleotide- Directed Mutagenesis 1. Target gene is cloned into a plasmid vector and dispensed into 2 tubes. 2. Two primers are added to each tube. One primer (e.g. 1 and 3) is completely complementary to a sequence within or adjacent to the cloned gene except for one nt. The primers (1 and 3) w/ the nt change anneal to opposite strands so both nt of a specific base pair are targeted. 16

17 3. After PCR, linear DNA is synthesized, how to join both ends? The positioning of the hybridization regions of the primers is such that, after PCR, the amplified DNA have different ends. different positioning of the ends, a strand (e.g. 1) from one tube hybridizes w/ its complementary strand (e.g. 3) from the other reaction tube to form circular DNA w/ two nicks. This procedure introduces a specific point mutation into a cloned gene w/o the need to insert the cloned gene to M13. Random Mutagenesis with Degenerate Oligonucleotide Primers What happens in reality is that usually which a.a. needs to be modified is unknown generate all the possible a.a. changes at one site. Ex: chemical synthesis of oligo primers w/ any of the 4 nt at defined positions. 17

18 The soln containing G also contains a few % of other 3, the resultant primer is a heterogeneous set, which will generate a series of mutations that are clustered in a defined portion of the target gene. Two advantages: 1. No need to know the exact role of a particular a.a. in the function of the protein. 2. Unexpected mutant proteins w/ novel and useful properties may be generated, the introduced changes are not limited to one a.a. * if the desired properties are not found repeat the procedures w/ a set of primers that s complementary to a different region. Protein Engineering 20 of the many thousand enzymes account for 90 % of the enzymes currently used in industry. Why are other enzymes not used? Their activities evolved from natural conditions are not well suited for in vitro functions in industry (e.g. high T, high P). Although thermo-tolerant enzyme can be isolated from thermophilic microorganism, these organisms often lack the particular enzyme is required protein engineering by directed mutagenesis and gene cloning. Ex: Adding S-S bonds (between cysteine CH 2 -SH) the stability (may not unfold readily resistance to organic solvents and extremes of ph ) Q: Does extra S-S perturb the normal function of a protein? 18

19 References: [1] Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K. Short protocols in molecular biology. New York: John Wiley & Sons, [2] Watson J, Gilman M, Witkowski, J Z, M. Recombinant DNA. New York: W.H. Freeman and Co., [3] Glick B, Pasternak J. Molecular Biotechnology: Principles and Applications of Recombinant DNA. Washington, D.C.: ASM Press,