lab 9 Flashcards
polymerase chain reaction
a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment
comprised of three basic steps: denaturation of dsDNA template, primer
annealing, and primer extension, also called elongation
DNA polymerase
used to amplify a piece of DNA by in vitro enzymatic replication
enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA
primers), which are required for initiation of DNA synthesis
can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5’-3’ direction
taq polymerase
a heat-stable DNA polymerase
lacks a 3’ to 5’ exonuclease proofreading activity
makes DNA products that have A (Adenine) overhangs at their 3’ ends
DNA primers
DNA polymerase can add a nucleotide onto only a preexisting 3’-OH group, and, therefore, needs a primer at which it can add the first nucleotide
Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase
denaturing of DNA template
the dsDNA template is heated to around 95 degrees C, causing the strands to separate
primer annealing
the temperature is decreased to allow the primers to anneal to the separated DNA template strands
primer extension
elongation or creation of a new complementary strand of DNA for each strand for the dsDNA template occurs when the temperature is held at approximately 72 degrees C and nucleotides are added to the primer.
how to set up a pcr reaction
25 uL reaction
add DNA polymerase, DNA polymerase buffer, dNTP mix, primer 1, primer 2, DNA template, and dH2O
run through the pcr thermocycler (18 cycles)
major steps of pcr
denature, anneal, extend
thermal cycling
alternately heating and cooling the PCR sample to a
defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA
helicase
required to unwind DNA from a double strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication
proofreading
The 3’->5’ exonuclease activity of the enzyme allows the incorrect base pair to be excised
dNTP mix
the nucleoside triphosphates containing deoxyribose
