[LAB] Unit 4.1 PCR Flashcards
(35 cards)
2 big issues solved by PCR
Specificity
Amplification
The human genome is composed of how many bp?
3.4 B bp
E. coli is composed of how many bp?
4.6 M bp
T or F: There are a lot of other sequences in a genome that we’re not interested in detecting.
T
To be visible on an agarose gel, need around ____ DNA for fluorescent stain (or around 25ng for FastBlast).
10ng
Avogadro’s number
6.02e23
PCR was developed by _____ ______ in _____
Kary Mullis; mid-1980s
Mullis was granted a Nobel prize in chemistry in ____
1993
PCR is also known as a “_____ machine” for DNA
copy
T or F: The PCR is a simple technique developed in 1985 to amplify sequence-specific DNA fragments in vivo.
F
in vitro
T or F: PCR can be performed in as fast as 1 hour
T
PCR amplification is achieved by using ________ primers.
oligonucleotide
These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.
Oligonucleotide primers
The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the ________.
template
T or F: The new strands have defined 5’ ends (the 5’ ends of the oligonucleotide primers), whereas the 3’ ends are potentially ambiguous in length.
T
T or F: During PCR, the DNA Polymerase is used as a template to synthesize a new DNA strand.
F
the existing DNA molecule
This causes strands to separate
Heat
Exact-length target product is made in the ___ cycle
3rd
T or F: Low [Mg2+] increases specificity
T
↓ Mg2+; ↑ Specificity; ↓ Yield
T or F: High [Mg2+] stabilizes primer annealing, can increase sensitivity, can decrease primer specificity
T
↑ Mg2+; ↑ Sensitivity; ↓ Specificity
Effect of too little DNA Polymerase
Insufficient product
Effect of too much DNA Polymerase
decreased specificity
2 primers anneal each other instead to the intended DNA sequence
Primer-dimers
Taq Pol activity decreases above ____
93 C