Polymerase Chain Reaction Flashcards
What does genetic engineering allow you to do
To isolate, identify, analyse, modify, transfer single genes of a genome of an organism
What are the 2 approaches for studying specific DNA sequences
1- To detect a specific sequence and then use molecular hybridization and then label a probe that will attach to specific sequences and then have a way to find the probes which have attached.
2- To amplify a certain sequence of DNA using cloning then you can use either a cell based or a cell free cloning
How does cell based cloning work
You need a host cell, a vector, a way of attaching the 2 types of DNA together, and screening to identify transformed complexes
How does cell free cloning work
By PCR, using a thermo polymerase and by denaturation, annealing and extension. No cells are needed, it can be done in a tube
How is molecular cloning done
Insert a DNA fragment into a self replicating vector and this will replicate. The required sequence elements will always include an origin of replication and a selectable marker
The PCR allows us to
Amplify a specific DNA sequence from a complex sample in vitro, the genes can be cloned without need to make DNA libraries
Initially how was PCR done
By hand, using 3 water tubs, and test tubes, and each time we feature there has to be DNA polymerase added
How is PCR done now
The DNA polymerase was changed to Taq DNA polymerase which is heat resistant, a machine called the thermal cycler was used to change the temperature
Define molecular cloning
insertion of a DNA fragment into a self replicating vector and its subsequent propagation, needs an origin of replication and a selective marker
what is a cloning vector
a vehicle for carrying foreign DNA into a suitable host cell, vectors are self replicating DNA molecules into which exogenous DNA can be inserted
Tap DNA polymerase is
the first isolated thermostable DNA polymerase, optimal temp was 75-80
what are advantages of RT-PCR over other RNA analysis techniques
speed
efficiency
specificity
sensitivity
Applications of RT-PCR
gene expression analysis, RNA sequencing analysis, Diagnosis of infectious diseases, diagnosis of genetic diseases, identification of intragenic translocations
What are the 2 ways in which we choose primers so that it doesn’t interfere with DNA amplification
~choose primers annealing to different exons
~choose primers annealing across an exon-exon junction
list PCR applications
microsatellite amplification, DNA fingerprinting, paternity testing, genetic testing, diagnosis of genetic diseases, prenatal diagnosis, identification of retroviral infection, tumor diagnosis, identify transgenic crops,
