Unit 2: The polymerase chain reaction Flashcards
What is PCR ?
A method used to amplify large amounts of DNA from a small amount of starting material
What type of DNA polymerase is used in PCR ?
Taq DNA polymerase
What is Taq made from ?
Bacteria that lives at high temps in hot springs and deep sea vents
What is an amplicon ?
A piece of DNA that is the product of amplification
What is a forward strand ?
5’ to 3’
What is a reverse strand ?
3’ to 5’
Explain the process of PCR (4)
1) Heat to 94 degrees to denature DNA strands
2) Cool to 50-60 degrees to allow primers to anneal
3) Heat to 72 to allow for elongation of new strands (Addition of nucleotides)
4) 2 New strands of DNA generated (At end of first cycle)
What is an upstream primer ?
Short sequence of nucleotides used to initiate the amplification of a specific DNA segment
What is the reverse primer in respect to the reverse strand ?
The reverse primer is complementary to the reverse strand but it needs to be written as 5’ to 3’ so it is reversed
Can primers also anneal to products of the PCR ?
Yes they can
How many cycles of PCR are there typically ?
30-40 cycles
What is included in a PCR reaction mix (7) ?
1) dNTPs
2) Reaction buffer containing Mg2+ ions
3) Forward primer
4) Reverse primer
5) Taq polymerase
6) Template DNA
7) dH2O
What are 5 things to consider when designing a primer for PCR ?
1) 18-22 nucleotides in length
2) GC content of 40-60% (More GC to AT)
3) Tm (melting temp) of 55-65C
4) Both primers should have a similar Tm
5) Primers should ONLY have similarity to site being amplified
How long is the initial denaturation stage ?
2-5 mins
How long is the denaturation stage ?
30-60 seconds
How long is the annealing stage ?
30-60 seconds
How long is the elongation stage ?
30-60 seconds
How long is the final elongation stage ?
5 mins
What 3 sections of the PCR programme needs to be repeated 25-35 times ?
- Denaturation
- Annealing
- Elongation
How can the total number of DNA produced be calculated ?
2^ number of cycles
How can the amplification of DNA in PCR be described ?
As being exponential
What are the 2 stages of PCR ?
- Exponential phase
- Plateau phase
What is the exponential phase ?
100% PCR efficiency- amount of DNA doubles each cycle
What is the plateau phase ?
PCR is less than 100% efficient
Why does the actual yield from a PCR plateau not carry on as theoretical suggests ?
- Damages Taq and used up available dNTPs and primers
- Enzyme degrades
- Run out of reagents and nucleotides
How can PCR products be analysed ?
By gel electrophoresis
What is one problem with Taq polymerase ?
It lacks 3’-5’ exonuclease proofreading activity so it can’t reverse and correct errors if it incorporates the wrong base
What are 4 uses of PCR ?
1) Amplification for cloning
2) Amplification for DNA sequencing
3) Detection of mutations
4) Detection of relatedness
What is RT-PCR ?
Reverse transcription PCR
What is RT-PCR ?
- Allows for detection of gene expression in cells and tissues
- Copy of mRNA is made into cDNA
- cDNA is used as template strand
What 3 primers are used in RT-PCR ?
1) Gene specific
2) Oligo dT
3) Random hexamers
Why is RT-PCR used in virus testing ?
- They are highly sensitive so can detect copies 17 days after infection
What is qPCR ?
Real time or quantitative PCR
What is the function of qPCR ?
It allows us to accurately determine the number of copies of a DNA sequence in starting material
What is SYBR ?
A green dye that only binds to double stranded DNA
What is the SYBR green method ?
- As the amount of PCR product increases each cycle so does the amount of SYBR green dye bound
- Laser is fired into sample and floresence is recorded
What are 4 uses of qPCR ?
1) Quantative measurement of gene expression between samples
2) Analysis of copy number variations
3) Quantification of virus numbers in an individual or sample
4) Microbiological monitering of food/water
