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
qPCR
Quantitative or Real-Time PCR
Quantitative or Real-Time PCR functions
Quantitative - determines amount of PCR product generated
Real-time - quantify reactions in “real-time”, as it happens
Quantitative or Real-Time PCR process
Each reaction tube contains labelled DNA/probe that emits fluorescent light when illuminated by laser beam
Correlation between light emitted and amount of PCR product
- SYBR Green
- TaqMan probes
qPCR – Taqman probe
- Complementary to a region in the amplicon
- Labelled with a “reporter” (R) at 5’-end of probe (e.g. FAM)
- Labelled with a “quencher” (Q) at 3’-end of probe (e.g. BHQ)
When probe is intact, Q interferes with fluorescent light emitted by R, no fluorescence detected
During extension, Taq polymerase cleaves R from Q - releases fluorescent light when excited by laser
Fluorescence detected and quantified electronically
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.
The real-time PCR is one of the most valuable modern PCR techniques
Using the premise that the amount of cDNA one can produce is dependent on the number of mRNA molecules present to start with in your sample.
The real time PCR enables one to quantify amplification reactions (i.e. how many copies of an mRNA transcript do I start with) unlike RT-PCR which gives you a relative view of mRNA abundance.
While a real time PCR relies on all the components of a normal PCR, it introduces a fluorescent probe into the reaction.
The real-time PCR probe works through fluorescent quenching
Two commonly used probe for the real-time PCR are a dye called SYBR® Green and TaqMan® probes.
SYBR Green is a dye which binds dsDNA, so as more DNA is produced from cDNA (during the PCR) more dye will bind the dsDNA target amplified by the primer sets.
The Taq-Man probe is complementary to specific regions of the target DNA between where the forward and reverse primers for PCR bind.
Increases in DNA copy number are reflected in increases in fluorescent light, as the TaqMan probe is incorporated into the growing number of molecules of the target DNA.
