PCR- polymerase chain reaction Flashcards
who developed the PCR technique
Kary Mullis, he was awarded the 1993 Nobel Prize in Chemistry
What is a PCR reaction?
Copying a specific DNA sequence through a series of in vitro reactions can amplify target DNA sequences that are initially present in very small quantities in a population of other DNA molecules.
[ Rapid method of obtaining multiple and specific copies of a particular region of DNA - No cloning and/or host cells required]
One needs to know some basic information of your target sequence.
This information is used to design two ________________.
The primers are short (usually 20 nucleotides) ___________.
One primer is _____________ to the 5’ end, the other to the 3’ end of the target sequence.
oligonucleotide primers
ssDNA sequences
complementary
The components of a PCR reaction
Target dsDNA
DNA Polymerase (Taq polymerase – thermostable)
Primers (Fwd & Rev)
dNTPs
Mg+2 in buffer solution - as cofactor for Taq
The fundamental workings of a PCR reaction rely on __________where, ______________________________. This process is ____________.
thermal cycling
DNA is denatured, primers bind and Taq polymerase then copies the template
exponential amplification
5 - 30 cycles of:
Denaturation: ~94°C ~1min
Annealing: 45 - 65°C ~30sec+
Extension: ~72°C ~1min
PCR – One cycle of amplification occurs in 3 steps per cycle
1.) Denature DNA (92-95 C)
- Start with a piece double-stranded DNA
–> contains target sequence you want to amplify
- Denature dsDNA using temps between 92-95 C
2.) Anneal primers (45-65 C)
- oligonucleotide primers that have been designed to amplify the target sequence bind to the sense strand and anti-sense strand of the denatured dsDNA
3.) Extend primers (65-75 C)
- Taq polymerase uses the primer and the template DNA to begin extension in the sense and anti-sense direction
- This creates 2 duplex DNA molecules
One cycle of amplification _______ the number of DNA molecules
doubles
Three cycles of amplification
- In the first cycle of a PCR the target DNA sequence is doubled.
However, note that the target DNA is still associated to the template DNA. - After three cycles, the target DNA begins to appear as a dsDNA product that is not associated to the template DNA.
- Beyond the third cycle the process of copying the target becomes exponential
PCR – advantages over cloning
Rapid (few hours)
Very sensitive & specific (down to single cell)
Effective with poor templates (degraded, embedded, contaminated)
Basis for many downstream applications
LIMITATIONS to PCR
Require knowledge of flanking sequences (for primers)
Sensitivity = contamination issue (complex)
Not for long segments of DNA (up to few 1000 nts)
RT-PCR
Reverse Transcription PCR
Reverse Transcription PCR function
For studying gene expression
Reverse Transcription PCR process
RNA extracted from tissue/cells of interest
Use reverse transcriptase (RT) to convert RNA to ds-cDNA
PCR amplification using fwd and rev primers
The reverse transcription PCR (RT-PCR) can be used for studying gene expression
Gene expression in this context means the transcript abundance of a gene, when the gene is activated or de-activated.
We have already learnt that the enzyme reverse transcriptase can create cDNA from mRNA and, if we couple this concept to a PCR reaction - we have a powerful molecular tool.
Because the amount of cDNA produced by reverse transcriptase is linked to the abundance of the mRNA molecules present at a specific time, one can use the RT-PCR to determine the relative expression of a gene in different samples.
In the example Gene A is expressed only in normal cells (two molecules of mRNA shown) but, not in cancer cells. So if one makes cDNA from normal cells vs. cancer cells, a PCR reaction on the cDNA using primers for Gene A will yield an amplicon only in cDNA from normal cells. This would tell us that Gene A is ONLY EXPRESSED in normal cells.
