🧆 🌊 ®️ Flashcards

1
Q

Polymerase chain reaction (PCR)

A

amplifies a small amount of DNA into large amounts

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2
Q

What is the name of the sample of DNA needing to be copied

A

The template DNA (often a known sequence or gene)

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3
Q

Template DNA

A

Typically double-stranded and only extremely small quantities are sufficient

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4
Q

What are the reagents needed for PCR

A
  • a pair of oligonucleotide primers
  • supply of nucleotide triphosphates
  • Taq DNA Polymerase (actually makes the copies)
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5
Q

What do the oligonucleotide primers have?

A

sequences complementary to the ends of the template DNA

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6
Q

Reverse transcriptase PCR (RT-PCR)

A

utilizes reverse transcriptase to make a cDNA copy of mRNA. PCR is later used to amplify the cDNA. This is useful as eukaryotic DNA tends to have long stretches of noncoding introns.

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7
Q

What are the two steps of RT-PCR?

A
  1. reverse transcriptase recognizes the 3’ end of primers containing repeated thymines and creates a DNA strand complementary to the RNA
  2. The RNA strand is replaced with another DNA strand -> double stranded cDNA that can be amplified.
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8
Q

What ends of the template strand do the primers anneal to?

A

5’ end of the sense strand and 3’ end of the antisense strand

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9
Q

What is the basic mechanism for PCR?

A

-denaturation of the template
-annealing of the primers
-making a complementary copy using DNA polymerase

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10
Q

Inverse PCR

A

Method for using PCR to amplify unknown sequences by circularizing the template molecule

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11
Q

What does the process of PCR require

A

changing of temperatures (in a cyclic manner)

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12
Q

what is the general sequence of inverse PCR?

A

DNA is cut with a RE that cuts upstream and downstream but not within it. The linear piece of DNA is circularized and amplified with primers that anneal into the known region. PCR products will have the unknowns that can be cloned.

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13
Q

degenerate primers

A

primer with several alternative bases at certain positions - they are based on amino acid sequences and contain different nucleotides at the wobble position

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14
Q

What are the modifications between the PCR cycle and DNA synthesis in vitro

A

-Double strand template is denatured with high heat (not enzymes)
-the temperature is lowered so primers anneal to their binding sites
-primers bind to opposite strands of the template (one at the beginning and one at the end)

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15
Q

What is the most widely used polymerase for PCR?

A

Taq polymerase (stable and does not denature at high temp.)

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16
Q

What is KEY to the PCR process?

A

The primers must anneal in the correct location

17
Q

What will happen if both primers anneal to the same strand?

A

The reaction will NOT work