Alternate PCR Flashcards
What can be changed in PCR?
Primers Polymerase Template Target Temperature cycle Direction
What types of PCR change the template and temperature?
Colony PCR
Fast cycling PCR
Describe colony PCR
DNA isn’t extracted from bacteria but the entire colony is added to the master mix
Performed using either a primer pair insert specific primers or plasmid specific primers that flank the insert
Used for rapid identification of correct ligation and insertion of inserted DNA in a plasmid
Describe fast-cycling PCR
Allows amplification of specific PCR products with significantly reduced cycling time
Buffer used increases the affinity of Taqp for short ssDNA thus reducing primer annealing time
2 step thermal profile- 94 denaturing and 68 annealing
What types of PCR change the polymerase?
High fidelity PCR
Hot-start PCR
Describe high fidelity PCR
Polymerase has a low error rate and significant binding affinity during amplification
Used in cloning, SNP analysis, and NGS applications
Describe hot-start PCR
Reduces occurrences of undesired products and formation of primer-dimers
Separate reagents until it reached denaturing temperature
Increased product yield
Takes less effort
Reduces risk of contamination
What types of PCR change the primers?
Multiplex PCR Amplified fragment length polymorphism (AFLP) PCR Intersequence specific PCR Assembly PCR Ligation mediated PCR Asymmetric PCR Inverse PCR Miniprimer PCR Nested PCR
Describe multiplex PCR
Multiple targets in one run
All primer pairs need similar annealing temperatures
Cheaper and quicker
Used in genotyping, mutation and polymorphism analysis, and pathogen detection
Describe AFLP PCR
Uses selective amplification of digested DNA fragments to generate unique fingerprints for genomes of interest
Generates large numbers of marker fragments without prior knowledge of the genomic sequence
Uses restriction enzymes and allows attachment of adaptors to sticky ends of fragments
Amplification using primers that are complementary to the adaptors
Then use agarose gel electrophoresis
Used to identify closely related species
Describe intersequence specific PCR
DNA fingerprinting that uses primers selected from specific segments repeated throughout a genome
Uses microsatellite sequences that are 16-25 bp long as primers in a single primer PCR reaction
Describe assembly PCR
Assembly of large DNA oligonucleotides from multiple shorter fragments
Used to make novel genes
Describe ligation-mediated PCR
Allows amplification without knowing the sequence of all the priming sites
Uses adaptors that are initially ligated to fragments of the target DNA
Primers the bind to the linker sequences
Describe asymmetric PCR
Used to preferentially amplify 1 strand of dsDNA
Achieved using an abundance of 1 primer
Describe inverse PCR
Used to amplify DNA when only limited sequence information is available
Restriction enzyme digestion, ligation
Results in a looped fragment which can act as a primer
Primers point away from each other
