PCR Flashcards
What are the three steps of PCR?
Denaturing, Annealing, Extension
If you only use one primer for PCR will it result in amplification?
Only DNA replication will occur, 30 copies will be made
At what temperatures do all steps occur at?
denaturing-95
annealing - 55
extension - 72
What types of DNA can be used in PCR?
linear, plasmid or cDNA
What are some of the important factors to consider when designing PCR primers?
length- 17-25 nucs, longer primers have greater specificity
temperature: too high-no anneal, too low-mismatches
What are some problems that occur with the specificity of primers?
If primers have complementary ends, they can bind together, and if you have self-complementary sequences within a primer you can form hairpin structures
What are the comparisons of Taq polymerase, Pfu polymerase and Phusion polymerase?
Taq - stability up to 95 degrees, faster extension, adds adenosine extension and has 3’ - 5’ exonuclease activity which gives it proof-reading ability
Pfu - lower error rate, still have 3’-5’ exonuclease activity, can amplify longer fragments
Phusion- just modified Pfu-like enzyme, much faster, error rate generally similar to Pfu, and can amplify longer DNA fragments
What are the purpose of additives in PCR and give examples?
Increase efficiency and reduce mispriming
Glycerol-enzyme stabilizer, Dimethyl sulfoxide-ease through secondary structures
What are the two major issues with PCR?
Contamination and non-specific amplifications
What are the steps to creating a TA vector?
- Treat blunt end cut vector with Taq in the presence of dTTP
- Engineered a vector with Xcml site
What is the process of cloning by restriction sites?
New restriction sites are added to the end of the 5’ end of primers and can then be easily spliced for easier insert into other vectors
Describe the process of cloning without restriction sites.
You need to have two DNA sequences with overlaps, T5 exonuclease remove nucleotides from the 5’ end of the molecule, Phusion fills in the gaps and Taq ligase joins the products together
Describe Allele-Specific PCR
This introduces a single mismatch into the allele, the mismatch will produce a bump and Taq cannot extend off of that, the 3’ exonuclease will allow for the removal of this. Only one allele of the two will be changed, the one primer must be a perfect match
application- 23 and me
Describe Long PCR
This is specific for long genes that need to be amplified, use Pfu
Describe Assembly PCR.
You are combining two PCR fragments into one. You will have 4 primers in total, 2 of them need to overlap to properly anneal
Describe RT PCR
take mRNA and turn it into cDNA, using a primer that is complementary to the poly A tail, only one strand present so only one primer, can lead to non specific amplification
Describe multiplex PCR
To test for differences in wild type and mutant genes, two different sets of primers used
Describe Inverse PCR.
Amplifies and obtains DNA of unknown sequence, first DNA is digested with an RE and ligated to create cDNA, primers are designed to extend outwards from known sequences. Known sequence is in the middle of the two unknown.
Describe Bridge PCR
Primers are covalently linked to a solid surface, the template will bind to the primers and extend, the template DNA is washed away in the next step. The second cycle, the present DNA in solution will bind to adaptor and create a “bridge-like” formation, the reverse strand is produced and is denatured and the process is repeated
Describe Emulsion PCR.
This is a way to carry out 100s or 1000s of reactions instead of just one. In the oil phase emulsion, one droplet of water will have PCR reaction. The oil is treated with a surfactant. The product can then be extracted because primers are attached to solid particle
