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MOLBIO LEC MIDTERMS > POLYMERASE CHAIN REACTION > Flashcards

POLYMERASE CHAIN REACTION Flashcards

1
Q

THE HUMAN GENOME PROJECT
Started in: ___
Goal: ___

A

Started in 1990
Goal: Fully sequence the entire
human genome

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2
Q

– the genome is LARGE

A

SPECIFICITY

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3
Q

– amount of DNA samples are VERY small

A

AMPLIFICATION

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4
Q

Came upon a way to double his test target DNA by giving him 21 copies

Repeating N times would yield: 2N

A

Dr. Kary Mullis

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5
Q

Double-stranded DNA is separated into 2 single strands

what PCR step?

A

Denaturation

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6
Q

Denaturation
Temperature:

A

94-96oC

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7
Q

MOST CRITICAL step for specificity
* Primers hybridize to their
complementary DNA sequences

A

Annealing

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8
Q

Annealing
Temperature:

A

50-70oC

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9
Q

Melting Temperature (Tm) formula?

A

𝑇𝑚 = 81.5 deg 𝐶 + 0.41(%𝐺𝐶) − (675/𝑛)

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10
Q

Temperature at which ½ of the double-stranded DNA exist as single-stranded DNA

Affected by:

A

(1) Reaction conditions, (2) Salt concentration, (3) Mismatches,
(4) Template condition, (5) Secondary structure

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11
Q

Performed by DNA polymerase

A

Extension

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12
Q

Extension

  • Catalyzes formation of the
    ___ between dNTPs and the 3’ end of the primer
  • Temperature: ___
A

phosphodiester bonds (1)
68-72oC (2)

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13
Q
  • Single-stranded DNA fragments
  • 20-30 bp long
A

PRIMERS

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14
Q
  • 3’ to the sequences amplified
  • Hybridizes with the plus strand
A

REVERSE PRIMER

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15
Q

5’ to the sequences amplified
* Hybridizes with the minus strand

A

FORWARD PRIMER

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16
Q

T OR F?
Melting temp (Tm) of the forward and reverse primer must be different

A

F : MUST BE SIMILAR

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17
Q

NA Template
Routine clinical analyses requires:

A

100 ng – 1 μg

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18
Q

Building blocks of DNA

A

DEOXYRIBONUCLEOTIDE BASES

19
Q
  • Thermus aquaticus
  • Thermostable
  • Good fidelity
A

Taq polymerase

20
Q
  • Thermus thermophilus
  • Also has reverse transcriptase
    activity
  • Used in RT-PCR (RNA template)
A

Tth polymerase

21
Q

Provide optimal conditions for enzyme activity

A

PCR BUFFER

22
Q

Provides pH for optimal
enzyme activity and accurate
amplification

What am i and what pH???

A

Tris buffer (1)
pH 8 – 9.5 (2)

23
Q

Affect denaturing and
annealing temperatures

A

Monovalent CATIONS
(KCl and (NH4 )2SO4)

24
Q

T OR F?
↑ salt concentration = shorter
DNA sequences denature
slower

A

F : longer

25
Q

(MgCl2) what cations?

A

divalent CATIONS

26
Q

Rapidly & automatically change to the required temperatures for each step and holds it there for designated periods

A

Machine: thermal cycler

27
Q
  • Negative control without DNA
  • “Contamination” control
A

Reagent Blank

28
Q
  • Negative control with lacking the target sequence
A

Negative template

29
Q

✓ Enzyme is active
✓ Buffer is optimal
✓ Primers are priming
✓ Machine is working

A

Positive

30
Q

Major cause of contamination:

A

presence of PCR products from previous amplifications

31
Q

Induces base damage that catalyzes the formation of single- and double-stranded breaks in DNA

A

PHYSICal:
UV Light

32
Q

PHYSICal: UV Light

Enhanced effectivity with the addition of ___

A

psoralens

33
Q

Widely used method for
decontamination and workspace prep

A

CHEMICAL:
10% Bleach

34
Q

Substitutes the dTTP for dUTP in the PCR reagent master mix

A

Chemical:
dUTP-UNG system

35
Q

Chemical: dUTP-UNG system

___ degrades any nucleic acid containing uracil

A

Uracil-N-glycosylase (UNG)

36
Q
  • Carries the primer sequence
    and becomes a target for the
    next amplifications
A

MISPRIMES

37
Q
  • Primers binding to each
    other
A

PRIMER DIMERS

38
Q

T OR F???
Primers can bind sequences other than their exact complements on the target (low stringency)

A

T

39
Q
  • Resolving amplification products
  • Agarose digestion with β-agarase or with iodine incubation
A

AGAROSE GEL
ELECTROPHORESIS

40
Q

✓ Using spin columns
✓ Shrimp alkaline phosphatase
(SAP) + exonuclease I (ExoI)

A

Residual component
removal

41
Q

More than one primer pair added to PCR that are primed simultaneously

A

Multiplex PCR

42
Q

More than one primer pair added to PCR that are primed simultaneously

A

Nested PCR

43
Q

Starting material is RNA and
converted to DNA by reverse
transcriptase

Used in gene expression studies

A

Reverse transcriptase pcr
“RT-PCR”

44
Q

Detects how much of the target
sequence is present usually
by fluorescence

A

REAL-TIME PCR

MOLBIO LEC MIDTERMS (4 decks)

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