5 BIOC0005 EXAM QUESTIONS AND ANSWERS WITH
COMPLETE SOLUTIONS VERIFIED LATEST UPDATE
The Polymerase Chain Reaction (PCR)
method for "cloning in vitro", as opposed to cloning using E. coli
A simple and fast 'one-step' method for creating billions of copies of a target region of
DNA Billions of copies Amplify by PCR
PCR involves multiple cycles (up to ~35 cycles) of:
1- DNA denaturation @ 94oC - 98oC
2- Primers annealing to complementary DNA sequence @ ~ 50oC - 68oC
3- Elongation of primers by a thermostable DNA polymerase @ ~ 72oC
PCR made possible by: (3)
1. Thermostable DNA polymerases
2. Low-cost chemical synthesis of primers (oligonucleotides)
3. Programmable thermocyclers
PCR involves synthesis of a product of a specific size, defined by
the positions of the primers
This defined product is first made in the third cycle. From then on its number increases
exponentially (while the products of undefined size increase linearly)
after n cycles, we should have
,2n-2 molecules of product
Maximum rate is only possible during
exponential phase when substrates are unlimited and the enzyme is fully active.
In reality, even during this phase amplification is not at 2-fold/cycle, since correct
priming and complete strand synthesis is never 100% Once substrates run out or
enzyme 'dies' then reaction ceases.
Design considerations
Primer design
Length of amplicon
Annealing temperature
Primer design
• Optimum length is 18 - 24 bases (long enough for specificity, short enough for efficient
binding at annealing temperature)
• GC content 40-60% with, ideally, last two bases as G or C
(more likely to fold and stick = stronger.
3' bases G and C as polymerase needs 3' OH and end bound
firmly to template. Strong hold
• Avoid sequences that would allow primer-primer annealing
Polymerase can sequence from primers
• Tm of primers should be similar (<5 difference) and between 60-75C
Tm = theoretical temp where 50% primer molecules expect to anneal
Do you need to add additional sequence to the ends of the PCR product?
, • Can add 5' tail to primer, not involved in hybridisation, but end includes it
Length of amplicon
• Maximum length for successful PCR is ~10 kb
• But typical product size is 0.2 - 2.0 kb
• Smaller amplicons are amplified more readily (a certain % of DNA pol molecules will
fail to complete strand synthesis)
Annealing temperature
• Typically: Tm - 5oC (efficient annealing, but minimal 'off-target' binding)
• Gradient thermocyclers allow optimization higher annealing temp = more specific PCR
product
PCR applications:
1. detection and analysis
2. PCR for cloning
3. PCR for site-directed mutagenesis
4. Reverse Transcription-PCR (RT-PCR)
5. Quantitative PCR (qPCR)
PCR evolution
thin walled tubes
heated lid
COMPLETE SOLUTIONS VERIFIED LATEST UPDATE
The Polymerase Chain Reaction (PCR)
method for "cloning in vitro", as opposed to cloning using E. coli
A simple and fast 'one-step' method for creating billions of copies of a target region of
DNA Billions of copies Amplify by PCR
PCR involves multiple cycles (up to ~35 cycles) of:
1- DNA denaturation @ 94oC - 98oC
2- Primers annealing to complementary DNA sequence @ ~ 50oC - 68oC
3- Elongation of primers by a thermostable DNA polymerase @ ~ 72oC
PCR made possible by: (3)
1. Thermostable DNA polymerases
2. Low-cost chemical synthesis of primers (oligonucleotides)
3. Programmable thermocyclers
PCR involves synthesis of a product of a specific size, defined by
the positions of the primers
This defined product is first made in the third cycle. From then on its number increases
exponentially (while the products of undefined size increase linearly)
after n cycles, we should have
,2n-2 molecules of product
Maximum rate is only possible during
exponential phase when substrates are unlimited and the enzyme is fully active.
In reality, even during this phase amplification is not at 2-fold/cycle, since correct
priming and complete strand synthesis is never 100% Once substrates run out or
enzyme 'dies' then reaction ceases.
Design considerations
Primer design
Length of amplicon
Annealing temperature
Primer design
• Optimum length is 18 - 24 bases (long enough for specificity, short enough for efficient
binding at annealing temperature)
• GC content 40-60% with, ideally, last two bases as G or C
(more likely to fold and stick = stronger.
3' bases G and C as polymerase needs 3' OH and end bound
firmly to template. Strong hold
• Avoid sequences that would allow primer-primer annealing
Polymerase can sequence from primers
• Tm of primers should be similar (<5 difference) and between 60-75C
Tm = theoretical temp where 50% primer molecules expect to anneal
Do you need to add additional sequence to the ends of the PCR product?
, • Can add 5' tail to primer, not involved in hybridisation, but end includes it
Length of amplicon
• Maximum length for successful PCR is ~10 kb
• But typical product size is 0.2 - 2.0 kb
• Smaller amplicons are amplified more readily (a certain % of DNA pol molecules will
fail to complete strand synthesis)
Annealing temperature
• Typically: Tm - 5oC (efficient annealing, but minimal 'off-target' binding)
• Gradient thermocyclers allow optimization higher annealing temp = more specific PCR
product
PCR applications:
1. detection and analysis
2. PCR for cloning
3. PCR for site-directed mutagenesis
4. Reverse Transcription-PCR (RT-PCR)
5. Quantitative PCR (qPCR)
PCR evolution
thin walled tubes
heated lid
