PCR Biotek

7/27/2019 PCR Biotek

1/18

Lab 11: Polymerase Chain Reaction

What Is PCR?

The Polymerase Chain Reaction (PCR)

is a method developed by Kary Mullis in

the 1980s to make large numbers of

copies of a specific DNA sequence

starting from as little as a single molecule

of DNA


2/18


What Is PCR?

PCR can be thought of as an alternative to

cloning a fragment of DNA to produce

multiple copies.

,very specific information about the nature

of the sequence to be copied.


3/18


Important Ingredients for PCR

PCR is basically a form of DNA replication in

vitro.

This re uires:Template DNA (target to be amplified)

Oligonucleotide primers (relatively short,

single-strands of DNA)

Free deoxynucleotides (dNTPs)

DNA polymerase (Ex. - Taq polymerase

from T h e r m u s a q u a t i c u s )


4/18


Steps in the process

There are three basic steps in the PCR

process

These are:

ena ura on

Annealing

Extension


5/18


On cycle of PCR

Denaturation is usually the first step.

Double-stranded template DNA is separated

into single strands using high temperature

> 90o

C Second, the temperature is reduced (~50-

60oC) to allow the single-stranded primers

to anneal to complementary sequences in

the template DNA

Last, the temperature is raised (~72oC) toallow DNA polymerase to extend new

synthesis out from the primer.


6/18


These three steps are repeated for

- cyc es o ac eve anexponential amplification of the

desired sequence


7/18


8/18


PCR Advantages and Disadvantages

Advantages

Fast, simple, and specific

Need only small amounts of template for a

lar e am lification 2n

, n = number ofcycles)

Done i n v i t r o

Disadvantages

Requires some knowledge of sequence to

design primersSize limitations for target sequence

Prone to contamination


9/18


Additional applications for PCR

DNA fingerprinting

Possible to conduct genetic analysis of

single hairs, very small drops of blood, etc.

e ca agnoses

Valuable tool for many types of genetic

testing


10/18


11/18

PCR

Steps in PCR reaction

denaturation

separate parent

strands in preparation

new strand synthesis


12/18

PCR

annealing

stick primers to the

prime DNA synthesis

DNA synthesis

requires a primer to

start DNA synthesis


13/18

PCR

extension

addition of

nucleotides, one at a

time, to the growingend of the DNA strand

(3 end) using the

parent strand as the

template

cycle through 25-35times


14/18


15/18

PCR

Considerations incorrect PCR product size (primer design)

mis-priming oo low annealing temperature

redundant sequences

no PCR product no priming

oo high annealing temperature

primerdimers

com lementar se uence in rimers andheyll anneal

oo much template forgot enzyme forgot dNTPs oo much/too little MgCl2

PCR product with errors wrong temperature

annealing extension

oo much/too little MgCl2 affects polymerase proof reading

complex sequence GGG repeats

low fidelity enzyme Taq polymerase vs Pfu


16/18

RT-PCR reverse transcriptase-pcr

RNA containing virus

PCR doesnt work on

RNA templates

Extract RNA from virus/cells

RNA

make cDNA copy of

RNA sequence first

PCR the cDNA copy of

RNA


17/18

REAL TIME PCR

kinetic approach

early stages

www.biorad.com


18/18

PCR Biotek

Documents