polymerase chain reaction Flashcards
PCR is used for?
detecting and amplifying specific DNA sequences, screening hereditary and infectious diseases, cloning genes, and fingerprinting DNA
the first step of PCR is initialization, what happens?
the sample is heated to around 95C, which melts the hydrogen bond that hold the dsDNA together to create ssDNA
the second step of PCR is annealing, what happens?
the sample is cooled to around 55C, which allows the primers to anneal to the ssDNA
the third step of PCR is elongation, what happens?
the sample is heated to 72C, the primers are used as starting point for new complementary strands to form in the 5’ to 3’ direction using Taq DNA polymerase
the length of the elongation step depends on?
the length of the product DNA
reverse transcriptase-polymerase chain reaction (RT-PCR) is used to?
detect relative expression of specific gene products which is a gauge of gene transcription by measuring the relative amount of target mRNAs
in order to conduct RT-PCR, all the the mRNa must be?
isolated and converted into cDNA using the enzyme reverse transcriptase
in RT-PCR, once the cDNA is formed?
it is subjected to PCR
if the gene was actively transcribed then its mRNA will have yielded a cDNA which will be amplified by the PCR reaction and visualized on a gel
quantitative polymerase chain reaction (qPCR) is known as real-time PCR and uses?
a dye that is fluorescent and binds DNA or a fluorescent oligonucleotide probes which hybridizes to the sequence of interest
