Amplification Polymerase Chain Reaction • PCR – Polymerase Chain Reaction is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence. • PCR was developed in 1985 by Kary B. Mullis, who was awarded the 1993 Nobel Prize in chemistry for his work. • The act of copying DNA in the laboratory • Works similar to the way your own cells replicate their DNA • With nuclear DNA testing STRs (Short Tandem Repeats) are copied What are STRs? • Microsatellites called Short Tandem Repeats • Highly repetitive sequences • 2-6 bp repeat unit • Polymorphic regions of DNA – Number of repeat units differ between chromosomes as well as between individuals • polymorphisms may result from DNA recombination during meiosis • polymorphism may also be caused by replication slippage Fluorescent dye label AATG AATG AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Primer positions define PCR product size Why STRs are Preferred Genetic Markers • Rapid processing is attainable • Abundant throughout the genome • Highly variable within various populations • Small size range allows multiplex development • Allelic ladders simplify interpretation • PCR allows use of small amounts of DNA material • Small product size compatible with degraded DNA TPOX Positions of STR Markers D3S135 on Human Chromosomes 8 TH01 D8S1179 D5S818 VWA FGA D7S820 D2S1338 CSF1PO AME D13S31 L Sex-typing 7 D16S53 D19S43 D21S11 D18S51 Penta E 9 3 AME L Penta D PCR Amplification • Components of PCR amplification Master Mix – Template DNA (1-10ng) – Primers – Deoxynucleotide triphosphates dNTPs – MgCl2 – DNA polymerase – Buffer BSA Template DNA • The PCR sample may be single- or double-stranded DNA or RNA. • 0.5 -2ng of DNA is targeted • Approximately 20,000 target copies are optimal. • Preferential amplification can occur if target amount is too low. PCR Amplification • Components of PCR amplification Master Mix – Template DNA (1-10ng) – Primers (0.1-1.0 0mM) – Deoxynucleotide triphosphates dNTPs (200mM each) – MgCl2 (1.2-2.5mM) – DNA polymerase (0.5-5.00mM) – Buffer (Tris 10-50mM pH8.3, KCl 50mM) – BSA (100mg/mL) Primers • PCR primers are oligonucleotides hybridizing to opposite strands and flanking the region of interest in the target DNA. • Several variables must be taken into account when designing PCR Primers. • Primer Design Considerations: – Primer length – Melting Temperature (Tm) – Annealing temperature – Complementarity – Primer structure – Sequence content Primer Design • Primer length – typically 18-30 bases long to increase specificity and decrease cross- hybridization • Melting Temperature (Tm) – Measure the stability of the primer-template DNA. Temperatures in the range of 52-58°C produce better results. The Tm is affected by the primer length and GC content. – Tm differnece between primers should be less than 5°C in order to avoid preferntial amplification • Annealing Temperature (Ta) – Typically 5°C below the lowest Tm of the primers. Optimal Ta yields less non- specific amplification Primer Design • Complementarity – Self- complimentarity primers should be avoided.(no intra-primer homology beyond 4 base pairs) to prevent the formation of primer dimers called self- dimer. – Primers with complimentarity with other primers (especially at the 3’end) need to be avoided to prevent the formation of primer dimers called cross- dimers . Primer Design • Primer structure and sequence content – G/C Content • A 40%-60% G/C content is recommended for both primers. • Greater than 3 repeats of G or C at the 3’ end of the primer (GC clamps) should be avoided to prevent the formation of primer-dimers . – Primers with Secondary structure should be avoided – Primers with long runs of the same base should be avoided • this prevent “breathing” of the primer, and therefore causes mispriming Other Considerations for Primers • Degenerate primers • Fluorescently labeled primers Degenerate Primers • Point mutations at the primer binding locations may cause null alleles • Degenerate primers are a set of primers that have several options in the sequence allowing for point mutations • When you type a sample with one kit it will appear as a homozygote if a null allele is present, but when typed with degenerate primers the null allele may be revealed showing that it was truly a heterozygote. Methods for Fluorescently Labeling DNA • Intercalating Dyes (post-PCR) • Dye-labeled nucleotide insertion during PCR • Dye-labeled primer insertion during PCR Fluorescent Labeling of DNA with Primers • Dyes are attached to one primer in a pair used to amplify a STR marker • Dyes are coupled to oligonucleotides (primers) through NHS-esters and amine linkages on the 5’end of the primer: » Dye-(CH2)6-primer • Dye-labeled oligonucleotides are incorporated during multiplex PCR amplification giving a specific color “tag” to each PCR product • PCR products are distinguished using CCD imaging on the 310 PCR Amplification • Components of PCR amplification Master Mix – Template DNA – Primers – Deoxynucleotide triphosphates dNTPs (20 -200mM each) – MgCl2 – DNA polymerase – Buffer – BSA Deoxynucleotide Triphosphates • It is important to keep the four dNTPs (dATP, dTTP, dCTP and dGTP) in equal concentrations (20 µM-200 µM) to minimize mis- incorporated nucleotides. • Lowering the dNTP concentration can improve fidelity. PCR Amplification • Components of PCR amplification Master Mix – Template DNA – Primers – Deoxynucleotide triphosphates dNTPs – MgCl2 (0.5 -2.5mM) – DNA polymerase – Buffer – BSA MgCl2 • MgCl2 concentration may affect the following: – Primer annealing – Strand dissociation temperature of template – Strand dissociation temperature of PCR product – Product specificity – Formation of primer-dimers – Enzyme activity and fidelity MgCl2 • Generally, increasing the free magnesium concentration increases yield and decreases specificity and fidelity. • When using AmpliTaq® DNA Polymerase, too little free magnesium will result in little or no PCR product, and too much free magnesium ion may produce a variety of unwanted products and promote misincorporation. The optimal amount is in the range of 0.5 to 2.5mM. PCR Amplification • Components of PCR amplification Master Mix – Template DNA – Primers – Deoxynucleotide triphosphates dNTPs – MgCl2 – DNA polymerase (0.5-5.00mM) – Buffer – BSA AmpliTaq ® Gold - DNA Polymerase • Originally isolated from the thermophilic eubacterium, Thermus aquaticus. • It is now supplied as a recombinant enzyme from E. coli. • The enzyme is highly purified and is free of nonspecific endo- or exonucleases. • It is a highly processive 5'–3' DNA polymerase which lacks 3'– 5' exonuclease activity. • It is chemically modified to be inactive until the reactions reaches 95 oC for 10 or 11 minutes (Hot start PCR) which improves the specificity, sensitivity, and yield of PCR. • Allows for room temperature amplification set up. Hot Start PCR • Why Hot start PCR – Regular DNA polymerases exhibit some activity below their optimal temperature. Therefore during set up primers can anneal non-specifically to the template DNA at room temperature and non-specific products may result. Also at low temperatures primers may bind to other primers forming primer dimers which can be preferentially amplified over template DNA. With Hot start PCR the DNA polymerase does not show any activity at room temperature. So there is no extension of non specific products or primer dimers. PCR Amplification • Components of PCR amplification Master Mix – Template DNA – Primers – Deoxynucleotide triphosphates dNTPs – MgCl2 – DNA polymerase – Buffer (pH8.3) – BSA GeneAmp® PCR Buffers • The GeneAmp 10X PCR Buffer is composed of 500 mM potassium chloride, 100 mM Tris-HCl (pH 8.3 at room temperature), 15 mM magnesium chloride and 0.01% (w/v) gelatin (BSA). • The recommended dilution buffer for AmpliTaq® Gold DNA Polymerase has pH 8.3 at room temperature which will decrease to pH 6.9 when heated to 95oC. PCR Amplification • Components of PCR amplification Master Mix – Template DNA – Primers – Deoxynucleotide triphosphates dNTPs – MgCl2 – DNA polymerase – Buffer – BSA (100mg/mL) BSA and other Additatives In some cases, adding the following compounds can enhance the efficiency or specificity of PCR: Betaine (0.5–2 M) Bovine serum albumin (BSA; 100 ng) Detergents Dimethylsulfoxide (DMSO; 2–10%) (v/v) Gelatine Glycerol (1–5%) (v/v) Pyrophosphatase (0.001–0.1 units/reaction) Spermidine PCR Instrumentation • The instrument used to perform PCR is the thermal cycler. • Thermal cyclers allow for the rapid and accurate heating and cooling of DNA samples which is crucial to PCR. • Modern thermal cyclers have a heated lid this prevents PCR reagents from condensing on the top of the tube during the temperature cycling. PCR Cycling Process Step AmpFlSTR Kits PowerPlex Kits Initial incubation 95°C for 11 min 95°C for 11 min, 96°C for 1min Cycling 28 Cycles 32 Cycles Denaturation 94°C for 1 min 94°C for 30 secs (C1-10) 90°C for 30 secs (C11-32) Anneal 59°C for 1 min 60°C for 30 secs Extend 72°C for 1 min 70°C for 45 secs Final extension 60°C for 45 min 60°C for 30 min Final hold 4°C 4°C Polymerase Chain Reaction (PCR) Process 5’ 3’ 3’ 5’ Denaturation (94oC) 5’ 3’ 3’ 5’ 25 – 35 Annealing (59oC) cycles 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ Extension (72oC) 5’ 5’ 3’ 1 3’ 5’ 5’ PCR Process • Initial incubation – Performed at approximately 95°C for 11 min – Activates AmpliTaq ® Gold PCR Process • Denaturation (Performed approximately 94°C): – During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle). Incomplete denaturation allows for snapback to occur, which reduces product yield. PCR Process • Annealing (Performed at approximately 59°C) : – Ionic bonds are formed between the primers and the template DNA, the polymerase can attach and starts copying the template. The higher the temperature, the more specific binding occurs PCR Process • Extension (Performed at approximately 72°C) : – This is the ideal working temperature for the polymerase.Taq DNA Polymerase can add approximately 35 to 100 base pairs per second. – The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) PCR Process • Final Extension: – Performed at approximately 60°C for 45 min – Ensures complete adenylation – If there is excessive –A , an additional extension cycle can be performed if allowed by the laboratory SOPs. Polymerase Chain Reaction One copy of DNA One cycle Two copies of DNA Second cycle Four copies of DNA Third cycle Eight copies of DNA Cycles Cycle Number Copy Number 1 2 2 4 3 8 4 16 5 32 N 2n Controls Used in PCR • Two main controls used in PCR are a Positive and Negative control • Positive Control – DNA supplied by makers of the amplification kits whose concentration is known. This is used to ensure there are no problems with the PCR process whether it be from the reaction components or the instrument itself. • Negative control – Contains all the PCR reagents but no DNA. This is used to ensure that there is no contamination in the reagents or that none was introduced during amplification set up. Critical Factors to remember for successful amplification • Wear gloves. • Change gloves to prevent contamination. • Use aseptic technique. • Use only plugged tips. • Use only sterile DI water. • Taq is kept in the DNA Free freezer. When not in use it shall be kept in the freezer. Place back in freezer as soon as possible. • Protect the Primer from light while in use. The fluorescent dyes attached to the primers are light sensitive. • Master MIX should be prepared just before use • Dilutions and calculations for proper template amount should be performed prior to amplification set up. Critical Factors to remember for successful amplification • Tubes should be properly labeled. • Only one tube opened at a time • Work in a hood if possible, if not use a face mask and avoid talking, coughing or sneezing over tubes. • Any human DNA introduced into the tube will be amplified so if your DNA or DNA from someone else gets into tube it will be amplified. • Develop a method to keep track of where you are during the set up process so if a distraction occurs you can easily find where you stopped. This avoids missing tubes or double loading or putting wrong sample in wrong tube.
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