polymerase chain reaction pcr. pcr allows for amplification of a small piece of dna. some...
TRANSCRIPT
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?