PCR Basics

1. 2. 3. 4.

Purpose of PCR Overview Components of PCR Reaction Variables

Temperature Cycle Times and Numbers Primer Buffer Polymerase

5.

Experimental Notes

Polymerase Chain Reaction

Amplify large quantities of DNA ( g quantities) from small quantities (fg quantities) [billion fold amplification] Analyze single DNA fragments out of large complex mixture. [ Human genome mixture of 12 million 300bp fragments] Alter DNA sequence directed mutagenesis.

What is the Polymerase Chain Reaction?

Its a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene. It can be thought of as a molecular photocopier

How Powerful is PCR?

PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm.

The Invention of PCR

Did He Really Invent PCR?

The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: Kleppe et al. (1971) J. Mol. Biol. 56, 341346. Progress was limited by primer synthesis and polymerase purification issues. Mullis properly exploited amplification.

The Basics of PCR Cycling

3035 cycles each comprising: denaturation (95C), 30 sec. annealing (5560C), 30 sec. extension (72C), time depends on product size. Whats in the Reaction?

WHATS IN THE REACTION ?

Template DNA Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium ions, bovine serum albumin) Nucleotides (dNTPs) Primers DNA polymerase (usually Taq)

PCR In Detail
Denature, anneal, extend and repeat the cycle 30 to 35 times. How does the polymerase know to stop when it reaches the other primer? Most textbooks to not fully explain PCR.

How many copies?

No target products are made until the third cycle. The accumulation is not strictly a doubling at each cycle in the early phase. At 30 cycles there are 1,073,741,764 target copies (~1109). There are also 60 other DNA copies

Overview of PCR
1. Temperature Cycling Denaturation Annealing Extension 94 55 72

2. Every cycle DNA between primers is duplicated

PCR Amplification

Exponential Amplification

30 cycles --- 1 billion copies in theory

Components of PCR Reaction

Template DNA Flanking Primers Thermo-stable polymerase
Taq Polymerase
Thermus aquaticus

dNTP
(dATP, dTTP, dCTP, dGTP)

PCR Buffer (mg++) Thermocyler

PCR Variables
1. 2. 3. 4. 5. Temperature Cycle Times and Temps Primer Buffer Polymerase

Temperature
Denaturation
Trade off between denaturing DNA and not denaturing Taq Polymerase Taq half-life 40min at 95 , 10min at 97.5 95

Annealing
Trade off between efficient annealling and specificity 2-5 below Tm

Extension
Temperature optimum for Taq Polymerase 72

Cycle Times and Temps

Typical PCR Run
Step Time/Temp

1 2 3 4 5 6 7 8

3 min at 95 30 sec at 95 1 min at 55 2 min at 72 Go to step 2 - 29 times 8 min 72 0 min 4 End

How many cycles?

Increasing the cycle number above ~35 has little positive effect. The plateau occurs when: The reagents are depleted The products re-anneal The polymerase is damaged - Unwanted products accumulate

Primers
Paired flanking primers Length (17-28bp) GC content 50-60% GC Clamp Tms between 55-80 Avoid simple sequences e.g. strings of Gs Avoid primer self complementary
e.g. hairpins, homodimers, heterodimers

PCR Buffer
Basic Components
20mM Tris-HCL pH 8.4 50mM KCl 1.5 mM MgCl2

Magnesium Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote misincorporation. Potential Additives
Helix Destabilisers - useful when target DNA is high G/CWith NAs of high (G+C) content.
dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), urea formamide

Long Targets >1kb. Formamide and glycerol Low concentration of template: Polyethylene glycol (PEG)

PCR Polymerases
Taq, Vent, Pfu, others Native or Cloned Half-life
e.g. Taq 40 min half-life, Vent 7 hour half-life

3-5 Exo nuclease proofreading Fidelity (Error Rate)

Taq 1/10,000nt, Pfu 1/1,000,000

Processivity Extra bases at end

Notes
Typical Reaction
32.5 l 5 l 1 l 0.5 l 0.5 l 10 l 0.5 l 50 l dH2O 10 X PCR buffer + mg 200 M dNTP 50 M Left Primer 50 M Right Primer Worm or Fly Lysate Taq Pol (5 Units/ l) Total Vol

Master Mix
1 volume master mix 32.5 l dH2O 5 l 10 X PCR buffer 1 l 200 M dNTP 0.5 l Taq Pol (5 Units/ l) 39 l Total Volume
To set up 4 reactions prepare 4.4 volumes of reaction master mix 143 l dH2O 22 l 10 X PCR buffer 4.4 l 200 M dNTP 2.2 l Taq Pol (5 Units/ l) Individual reactions - 39 l master mix - 0.5 l Left primer - 0.5 l Right primer - 10 l worm lysate

DNA Sequencing

DNA Sequencing
Two main methods 1. Sanger dideoxynucleotide chain termination method (Commonly used method) A. Manual method B. Automated method 2. Chemical cleavage method (Maxam and Gilbert method) Not used nowadays Use of the technique: Provides the order of the nucleotides in a given DNA

DNA Sequencing - Sanger Method

Sanger Method

DNA Sequencing - Sanger Method

Procedure for the Sanger dideoxy chain termination technique

DNA to be sequenced is cloned into a vector, adjacent to a primer site Four reactions are set up -- each containing one of four ddNTPs DNA is synthesized in the presence of the ddNTPs, giving rise to sets of DNA products representing all of the possible size fragmentsfor the unknown sequence The fragments are resolved by gel electrophoresis and the sequence is read up from the bottom of the gel by identifying the lane giving the next larger size fragment

Composition of a DNA sequencing reaction

dNTP dATP, dCTP, dGTP, dTTP One type of ddNTP in each type of reaction DNA polymerase (Taq polymerase can also be usedcycle sequencing) One primer Template DNA Labeling- Radioactive and non radioactive

Labeling methods
1. Labeling the nucleotides S35dNTP or P32 dNTP Or Fluorescent labeled dNTP 2. Labeling the primers S35dNTP or P32 dNTP Or Fluorescent labeled dNTP

For sequencing the DNA is denatured into single strands the primer is hybridized to the template strand DNA is synthesized using DNA polymerase