Reverse Transcription & PCR (RT-PCR) Flashcards
what is transcription ?
The process of synthesising an RNA transcript with the transfer of sequence information from a DNA template
what is DNA transcribed into ?
a complementary sequence of bases, called messenger ribonucleic acid (mRNA), which is then used for protein synthesis
what is DNA transcribed into ?
a complementary sequence of bases, called messenger ribonucleic acid (mRNA), which is then used for protein synthesis
The genetic code defines the relationship between…..
- the sequence of bases in the DNA (or RNA transcript)
- the sequence of amino acids in a protein
The genetic code defines the relationship between…..
- the sequence of bases in the DNA (or RNA transcript)
- the sequence of amino acids in a protein
The genetic code defines the relationship between…..
- the sequence of bases in the DNA (or RNA transcript)
- the sequence of amino acids in a protein
The genetic code defines the relationship between…..
- the sequence of bases in the DNA (or RNA transcript)
- the sequence of amino acids in a protein
What happens to DNA in reverese transcription ?
A copy of DNA from mRNA is made called cDNA
What happens to DNA in reverese transcription ?
A copy of DNA from mRNA is made called cDNA
What happens to DNA in reverese transcription ?
A copy of DNA from mRNA is made called cDNA
What happens to DNA in reverese transcription ?
A copy of DNA from mRNA is made called cDNA
what does PCR stand for ?
Polymerase Chain Reaction
what does PCR do ?
Method used to amplify a sequence of DNA using a pair of oligonucleotide primers, each complementary to one end of the DNA target sequence
what does PCR do ?
Method used to amplify a sequence of DNA using a pair of oligonucleotide primers, each complementary to one end of the DNA target sequence
Essential Components of PCR – DNA Template
- Template DNA that contains target sequence
- Genomic (gDNA) or Complementary DNA (cDNA) can be used
- ≤ 1µg of DNA is required
Essential Components of PCR – DNA Template
- Template DNA that contains target sequence
- Genomic (gDNA) or Complementary DNA (cDNA) can be used
- ≤ 1µg of DNA is required
Essential Components of PCR – DNA Polymerase
- A thermostable DNA Polymerase to catalyse template dependent synthesis of DNA
- Taq polymerase is the enzyme of choice
- Synthesise 9000 bases in < 10 seconds
- Taq lacks 5′ → 3′ proofreading function
- Misincorporation rate of 1 nucleotide in ≈ 9000 nucleotides
Essential Components of PCR – DNA Polymerase
- A thermostable DNA Polymerase to catalyse template dependent synthesis of DNA
- Taq polymerase is the enzyme of choice
- Synthesise 9000 bases in < 10 seconds
- Taq lacks 5′ → 3′ proofreading function
- Misincorporation rate of 1 nucleotide in ≈ 9000 nucleotides
Principles of PCR
- The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses
- PCR amplifies a specific region of a DNA strand
- amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size
Essential Components of PCR – Primers
- Pair of synthetic oligonucleotides to prime DNA synthesis
- Complementary to the 3‘ ends of each of the sense and anti-sense strands of the DNA target
- DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers there is no double-stranded initiation site at which the polymerase can bind
Essential Components of PCR – dNTPs
- dNTPs – Deoxynucleoside triphosphates
- Standard PCR contain equimolar amounts of dATP, dTTP, dGTP and dCTP
- In a PCR reaction containing Taq and 1.5 mM MgCl2 [dNTP] 200-250 µM for each dNTP
- In a 50 µl reaction this [dNTP] should allow synthesis of ≈ 6 – 6.5 µg DNA
Essential Components of PCR – Buffer Solution
- Buffer solution to maintain pH
- Tris-HCl pH between 8.3 and 8.8 at room temperature
- When incubated at 72°C pH of the reaction mixture drops to around 7.2
Essential Components of PCR – Divalent Cations
- All thermostable DNA polymerases require free divalent cations to:
- React with dNTPs to form complexes that are substrates for the Taq polymerase
- Stabilise the primer-template complexes
Essential Components of PCR – Divalent Cations
- All thermostable DNA polymerases require free divalent cations to:
- React with dNTPs to form complexes that are substrates for the Taq polymerase
- Stabilise the primer-template complexes
Essential Components of PCR – Monovalent Cations
- Standard PCR buffer contains 50 mM KCl
- Works well for amplification of segments of DNA > 500 bp in length
- Increasing [KCl] to ≈ 70 – 100 mM may improve yield of shorter DNA fragments
Essential Components of PCR – Monovalent Cations
- Standard PCR buffer contains 50 mM KCl
- Works well for amplification of segments of DNA > 500 bp in length
- Increasing [KCl] to ≈ 70 – 100 mM may improve yield of shorter DNA fragments
PCR Procedure
- Denaturation
- Annealing
- Extension/elongation
- Final Extension
- Hold
PCR Procedure
- Denaturation
- Annealing
- Extension/elongation
- Final Extension
- Hold
Denaturing
- First step in the cycling stage and consists of heating the reaction chamber to 94–98 °C for 20 – 30 seconds
This causes denaturation of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules
Annealing
reaction temperature is lowered to 50–65 °C for 20 – 40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates
A typical annealing temperature is about 3–5 °C below the Tm of the primers used
During this step, the polymerase binds to the primer-template hybrid and begins DNA formation
Extension
The temperature at this step depends on the DNA polymerase used; for Taq polymerase a temperature of 72 °C is commonly used
The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify
Usually between 30 – 60 seconds
PCR Procedure – Number of Cycles
- The processes of denaturation, annealing and elongation constitute a single cycle
- Multiple cycles are required to amplify the DNA target to millions of copies
- The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles
- A reaction set for 30 cycles results in 2 to the power of 30, copies of the original double-stranded DNA target region
