Polymerase Chain Reaction

Transcription

1 Polymerase Chain Reaction

2 Forensic Genotyping Forensic samples: Limited quantity of DNA Poor quality (degraded) Need to have a mechanism to amplify the amount of DNA in the sample Polymerase Chain Reaction (PCR) fits the needs of Forensics perfectly: Sensitive, rapid and not limited by quality

3 PCR 1. Separate the Double Helix 2. Bind primers (2) to sequence you want to replicate 3. DNA Polymerase copies between two primers 4. Rinse and Repeat 5. Copies DNA between two primers exponentially

4 PCR 1. Separate the Double Helix Heat 2. Bind primers (2) to sequence you want to replicate Add

5 PCR 3. DNA Polymerase copies between two primers Add DNA Pol. 4. Repeat Many Times

6 Thermal Cycles Three main steps of temperature: degrees C Denatures the double stranded DNA degrees C Primer annealing temperature degrees C DNA polymerase extension Polymerase reads the sequence between the primers to produce a copy

7 Exponential Growth Because DNA is double stranded Add two primers Each cycle will double the amount of DNA Exponentially copying the template Theoretically: 3 cycles = 2 copies 12 cycles = 1000 copies 22 cycles = 1 million copies 32 cycles = 1 billion copies

8 Forensic Kits Reagent kits are now available to laboratories Simply add DNA template to pre-mixed PCR reagents: DNA concentration is known (quantified) Concentration is equalized between samples Put mix + DNA through thermal amplification cycles get product

9 PCR Needs You must know the sequence you are trying to amplify Primers: One for each side, exact sequence correct Excess of four dntps: 4 nucleotides Heat insensitive DNA Polymerase: So that reagents can be heated and cooled repeatedly

10 Primers Primer is a short piece of DNA: 20 to 30 bases long Oligonucleotide Designed to match the sequence surrounding the template exactly Hybridize to the correct place in the genome based on complementary base pairing at the correct conditions Added to PCR reaction in high concentration

11 dntps deoxyribose Nucleotide TriPhosphate: Building blocks of new DNA strands DNA Polymerase adds the correct dntp based on complementary base pairing Just as a cell replicates it s own DNA during S phase Added to PCR reaction in extreme excess so that reaction will never run out

12 Heat Insensitive Polymerase Normal DNA Polymerases will be denatured when heated to the temperature denatures double stranded DNA Old Method: Added more polymerase after each 95 stage Now: Use Taq Polymerase Heat insensitive doesn t denature Isolated from bacteria that lives in hot springs

13 Other reagents Tris-HCl (or 10X Buffer) Buffers reaction to correct ph for Polymerase Magnesium Chloride Salts are cofactor for Polymerase DNA sample Primers Taq Polymerase dntps

14 Master Mix When setting up for many different samples More accurate to prepare a Master Mix that contains all reagents except DNA Then aliquot Master Mix into each tube Finally add DNA sample Improves: Accuracy of each component Reproducibility of method

15 Controls Negative Control Same master mix but with water or buffer No DNA sample Proves PCR reagents are not contaminated Positive Control Master mix plus DNA of known concentration Proves PCR reaction/conditions work Extraction Blank Controls for DNA extraction reagents

16 DNA Template Normally PCR uses 1-50 ng of template Forensic DNA samples often have very low concentration of DNA DNA below 100 pg (0.1 ng) will often show allele dropout When one allele doesn t amplify but other allele does giving wrong genotype Falsely homozygous

17 Thermal Cycling Conditions Also known as Amplification Conditions The temperatures and amount of time at each temp that PCR reaction is set at Vary widely depending on: Length of DNA to be amplified Primer s ideal annealing temp Exact Polymerase in use Quantity and quality of DNA sample

18 Thermal Cycling Conditions Usually have 25 to 35 cycles Example protocol: 95 for 5 minutes hot start 30 cycles: 94 for 30 seconds 60 for 30 seconds 72 for 1 minute 72 for 10 minutes final extension 4 degrees to keep products from reacting

19 Thermal Cyclers Machines that accurately and quickly change the temperature of the tubes PCR Machines Enter the program for your amplification conditions Precise and accurate heating and cooling Old Method: Move the tube between 3 hot water baths

20 Possible Problems with PCR Primer annealing non-specifically At room temperature primers can start to anneal to template at random Primer Dimers Primer anneals to other primer Avoid both these by using Hot Start Polymerase Choose a Polymerase that only works once reactions have been heated to 95

21 Designing Primers Most important step of PCR may be designing the primers Primers determine: Exactly what DNA sequence is copied PCR yield For successful PCR primers must be: Specific to one highly conserved sequence Possess similar annealing temps Not interact with each other (primer dimers)

22 Length Optimal Primers 18 to 30 bases Annealing Temp 55 to 72 degrees GC Content 40 to 60% Unique oligionucleotide sequence Less than 800 bases between two primers No runs of >4 of same base in a row No hairpins (secondary structure) No complementary sequence to other primer (primer dimer)

23 Primer Design Should always BLAST your primers Will examine if primers can anneal anywhere else in genome Programs exist to help choose: Primer Express Primer3 Important parameters to input: Product size Primer length Ideal annealing temp

24 Multiplexing Adding more than one set of primers in the same reaction Produces more than one PCR product while only running one reaction Saves on amount sample used Saves time and money PCR is ideally suited to be multiplexed As long as you optimize your reactions

25 Optimizing Multiplexing Must optimize multiplex reactions: Products are all of different lengths Labeled with different fluorescent dye Primers need similar annealing temps Primers must not interact with any of the other primers Most important to optimize: Primer sequences Concentration of each primer

26 Optimizing Multiplexing Our average multiplex was 9 different products per tube: Three different colored dyes In commercial PCR kits markers are multiplexed together Already optimized 13 markers per tube

27 Real Time PCR Analyzes the PCR product while PCR reaction is occurring Allows quantification of starting amount of DNA Two main methods: TaqMan Assay Measures amount of specific product SYBR green Measures amount of any DNA produced

28 TaqMan Assay Uses a probe that binds to center of DNA sequence you are amplifying Probe contains both: Reporter dye Quencher dye stops reporter from fluorescence Whenever two dyes break apart you see fluorescence of reporter dye Probe breaks during DNA polymerization

29 R Forward TaqMan primer 5 probe Q Reverse primer Polymerization and Strand Displacement Forward primer R Forward primer R Q 3 Probe Cleavage (release of reporter dye) Reverse primer Reverse primer Q Fluorescence occurs when reporter dye and quencher dye are no longer close Completion of Polymerization Figure 4.4, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press

30 Real Time PCR More DNA polymerization more signal Idea is the more DNA you started with, the faster your reaction will reach a specific concentration of DNA Because amplification during PCR can be calculated exactly per cycle Set an arbitrary threshold of DNA concentration fluorescence of probe See how many cycles it takes to reach

31 C T Cycle Threshold How many cycles it took for reaction to pass arbitrary DNA concentration Set fluorescence of probe Normalize fluorescence to adjust for background differences in tube C T or number of cycles Used to calculate amount of starting DNA

32 ΔR n Normalized Fluorescence threshold Linear product growth Exponential product growth Plateau Cycle Number a b c d e C T Negative control Standard curve e d c C T b a Log[DNA] Figure 4.5, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press

33 Contamination Easy to contaminate DNA sample Exactly because PCR is so great at amplifying small amounts of DNA Need to be very careful: Scientist performing PCR Police officer collecting evidence Everyone involved with DNA sample is often genotyped as well: Staff elimination database

34 Appropriate Guidelines Police Officers have handout: Forensic Scientists: Lab coats Fresh gloves at all times Facial masks and hair nets Precautions are necessary when working with miniscule amounts or degraded DNA

35 Protect Against Contamination Physically separate PCR set up areas from PCR products: Have two different benches Different pipettes, sterile water, etc Wear and change disposable gloves Use aerosol resistant pipette tips Change tips every single time! Irradiate benches and spray with alcohol or bleach between uses

36 Advantages of PCR Very small amount of DNA can be used DNA can be degraded Reactions can be multiplexed Non-human DNA contaminates are not a problem Because primers are human specific Commercial kits are available for labs Only have to add DNA sample

37 Disadvantages of PCR PCR Inhibitors may block amplification: Indigo dyes Hemoglobin Amplification may fail: Allele drop-out false homozygous Polymorphisms at primer binding site Contamination from other DNA sources: Easy to get contamination Difficult to separate/identify sources

38 What NOT to learn Skip all specifics: Names of polymerases Polymerase s ideal temperature or ph Names of thermal cyclers All of this has already changed Will change quickly Depends on budget and desires of lab

39 Any Questions? Read Chapter Five