Polymerase Chain Reaction PCR. PCR allows for amplification of a small piece of DNA. Some applications of PCR are in: –forensics (paternity testing, crimes)

Polymerase Chain Reaction

PCR

PCR

• PCR allows for amplification of a small piece of DNA.

• Some applications of PCR are in:– forensics (paternity testing, crimes)– identification of human remains

• Sometimes problems if DNA is degraded

– Study human ancestry– Cloning – introduced amplified DNA

into a vector

PCR • PCR requires a thermostable DNA polymerase • The most common type of DNA polymerase is

Taq polymerase

• 2 Oligonucleotides called primers are also included in the PCR reaction (match and bind to the target sequence)

• 4 deoxynucleotides- The subunits of DNA that are incorporated into the new DNA copies

PCR Components

• Template DNA• Nucleotides (dNTPS)• PCR buffer

• Magnesium chloride (MgCl22)

• Water• Forward and Reverse primers• (Taq) DNA polymerase

PCR machine

• The PCR mixture which contains DNA polymerase, buffer, deoxynucleotides, primers, and template, go through a cycle of varied temperatures

• 3 steps in a cycle -Denature-Anneal-Extend

Steps in PCR• Denature

-Temperature is raised to near boiling (92ºC)-Template DNA is separated into two strands

• Anneal -Temperature is lowered (50º-65ºC)-Allows the left and right primers to base pair to their complementary sequences-Primers purpose is to bracket the DNA region to be amplified

Steps in PCR

• Extend -Temperature is raised to 72ºC-Taq polymerase attaches at each priming site-Uses the nucleotides from the reaction mixture and attaches it to the growing strand; these nucleotides are complementary to the target sequence

PCR Animation

• http://www.maxanim.com/genetics/PCR/PCR.htm

DNA replication in vivo

• Topoisomerase• Helicase• Primase• Single stranded binding protein• DNA polymerase• DNA ligase

PCR Results

• These 3 steps of PCR result in exponential growth N =2t x (No) of the DNA template in the mixture

• It’s typically repeated for 20-40 cycles

• But the product amount reaches a maximum after about 40 cycles due to depletion of the reaction components

PCR sample problem

• If you start with 30 copies of a target sequence and perform PCR for 45 cycles, then how many copies of the target sequence will you have at the end of the PCR amplification?

PCR- Problems

• Polymerase Errors– Taq polymerase lacks 3’ – 5’

exonulcease activity

• Unwanted amplification of products -Contamination-Nonspecific primer annealing

Electrophoresis follows PCR

• Used to determine if your PCR worked

• Determine if your sequence was present– If it was not present, the primers

would not bind and you will not get a band.

Setting up a PCR Reaction• Special small tubes or strips• Have thin walls – efficiently transfer heat• Try to prevent contamination

– Tips with filters– Gloves– Often set up in hoods with UV lights

• Need to have negative controls• Set up reactions on ice – don’t want

nonspecific amplifications– For example, primers binding to themselves

Types of PCR

• Real time (quantitative PCR)

• Reverse transcription PCR

Real time (quantitative PCR)

• Conventional PCR– Not quantitative– PCR products analyzed at the end of

the PCR

• Real time PCR– Quantitative– Amount of DNA measured at each

cycle and input DNA is determined

Real-Time PCR

• This procedure uses a fluorescence dye that will bind the DNA.

• Can view the increase in the amount of DNA as it is amplified.

• As the PCR reaction progresses, more DNA is produced and more fluorescence is detected

• Determine how much produced by comparing to controls with a given amount of DNA

SYBR green is a fluorescence dye that binds DNA

Adapted from Biorad.com

Sybr green can be first detected at the threshold cycle

Can also use Ethidium bromide

• Ethidium bromide intercalates into the DNA

• Take DNA after different cycles and add ethidium bromide

• Compare to known standards

Reverse transcription PCR

• Often used in combination with Real Time PCR

• Start with RNA and then make complementary DNA (cDNA)

• What enzyme is used?