[LAB] Unit 4.1 PCR Flashcards

(35 cards)

1
Q

2 big issues solved by PCR

A

Specificity
Amplification

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2
Q

The human genome is composed of how many bp?

A

3.4 B bp

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3
Q

E. coli is composed of how many bp?

A

4.6 M bp

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4
Q

T or F: There are a lot of other sequences in a genome that we’re not interested in detecting.

A

T

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5
Q

To be visible on an agarose gel, need around ____ DNA for fluorescent stain (or around 25ng for FastBlast).

A

10ng

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6
Q

Avogadro’s number

A

6.02e23

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7
Q

PCR was developed by _____ ______ in _____

A

Kary Mullis; mid-1980s

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8
Q

Mullis was granted a Nobel prize in chemistry in ____

A

1993

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9
Q

PCR is also known as a “_____ machine” for DNA

A

copy

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10
Q

T or F: The PCR is a simple technique developed in 1985 to amplify sequence-specific DNA fragments in vivo.

A

F

in vitro

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11
Q

T or F: PCR can be performed in as fast as 1 hour

A

T

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12
Q

PCR amplification is achieved by using ________ primers.

A

oligonucleotide

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13
Q

These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.

A

Oligonucleotide primers

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14
Q

The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the ________.

A

template

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15
Q

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.

A

T

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16
Q

T or F: During PCR, the DNA Polymerase is used as a template to synthesize a new DNA strand.

A

F

the existing DNA molecule

17
Q

This causes strands to separate

18
Q

Exact-length target product is made in the ___ cycle

19
Q

T or F: Low [Mg2+] increases specificity

A

T
↓ Mg2+; ↑ Specificity; ↓ Yield

20
Q

T or F: High [Mg2+] stabilizes primer annealing, can increase sensitivity, can decrease primer specificity

A

T
↑ Mg2+; ↑ Sensitivity; ↓ Specificity

21
Q

Effect of too little DNA Polymerase

A

Insufficient product

22
Q

Effect of too much DNA Polymerase

A

decreased specificity

23
Q

2 primers anneal each other instead to the intended DNA sequence

A

Primer-dimers

24
Q

Taq Pol activity decreases above ____

25
Increase number of cycles if rxn has ____ initial target molecules
<103
26
Inhibit amplification of nucleic acids by PCR
PCR Inhibitors
27
Common PCR inhibitors
Heparin Urea Hemoglobin
28
A strand of short nucleic acid sequences that serves as a starting point for DNA synthesis
Primer
29
The polymerase starts replication at the ___-end of the primer, and copies the opposite strand.
3'-end
30
The longer the primer, the more chance that it is ______
unique
31
T or F: The shorter the primer, the higher melting/annealing temperature
F The longer
32
The Tm of the probe should be ______ higher than that of the primers
5-10°C
33
The presence of G or C bases within the last five bases from the 3' end of primers
GC Clamp
34
The length of primer has to be at least ___ bases to ensure uniqueness.
15
35