PCR Flashcards
Uses of PCR
To insert DNA fragments into bacterial plasmid, genetic cloning, and protein production methods.
Components of PCR
Template DNA, primers, thermostable enzyme, high fidelity enzymes, reaction buffer, a thermocycler and the four nucleotides
Template DNA
Must be high purity and fully intact
Primer
Short single stranded DNA sequence designed to bind to specific complementary DNA region serving as a starting point for DNA synthesis.
Thermostable enzyme
Does not denature in PCR temps, taq polymerase is used. (From thermis aquaticus)
High fidelity enzymes
Enzymes that correct errors during DNA synthesis in 3’ to 5’ direction. PFu is commonly used.
4 nucleotides
dATP, dTTP, dCTP, dGTP
PCR Buffer
Maintains optimal pH and contains Mg+ ions to prevent DNA fragmentation.
Thermocycler
Water bath device with programmable temperature changes at certain times
Factors in primer design
Length, temperature, structure and restriction site
Length of primers
18-25 nucleotides, longer primers increases accuracy but can make secondary structures.
Temperature of primers
Melting temperature Tm is where 50% of the primer anneals to the template. Primers should be within 2° of their Tm (55 to 65°C)
GC content of primers
Should be 40 to 60%, which gives optimal stability as hydrogen bonding increases.
Restriction sites in primers
Allows targeted incision of restriction enzyme, making the product ideal for incorporation into plasmids.
Primer dimers
When primers attach to each other instead of template DNA, causing the PCR efficiency and yield to dramatically decrease.
Typical PCR programme
- 96°C for 2mins to denatured double stranded DNA and activate DNA polymerase.
- 97°C for 15secs repeats separation of DNA strands every cycle.
- 57°C for 30secs allowing primers to bind rapidly to complementary sequences. (Based on Tm)
- 72° C polymerase synthesis new DNA by adding dNTPs. Allow 1min per kilobase here, typically 10 mins.
- Cooled to 4°C after all cycles for storage.
Number of times a PCR is cycled
10 to 30 times
Multiple cloning sites
Short DNA sequence in a plasmid containing several restriction enzyme recognition sites. Used to facilitate PCR DNA fragments into plasmids.
Location of MCS
Upstream of promoter region or downstream of a selectable marker region.
Example of MCS
Plasmid pUC19 contains MCS with lacZ gene, allowing for blue white screening.
Blue / white colony screening
Inserted DNA disrupts lacZ function, turning colonies white in the presence of X gal and IPT6. Blue colonies have failed insertion.
Where should restriction sites be added on primer
Near the 5’ end to facilitate cloning
