Polymerase Chain Reaction and Alu Elements Flashcards

1
Q

What are the steps in a recombinant DNA study?

A
  1. Amplify gene of interest.
  2. Insert gene into vector.
  3. Insert recombinant DNA into living cells to propagate it.
  4. Restriction endonucleases are used in the process.
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2
Q

What is PCR?

A

Polymerase chain reaction is a technology that allows specific DNA sequences to be amplified.

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3
Q

What are the three major steps of PCR?

A
  1. Denaturation
  2. Annealing
  3. Extension
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4
Q

Denaturation

A

target DNA is denatured and the DNA strands are separated.
1 min 94 degC

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5
Q

Annealing

A

2 short oligonucleotide primers are annealed to the denatured DNA. Primers are complementary to the two 3’ ends of the DNA to be amplified.
45 seconds 54 degC

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6
Q

Extension

A

DNA Polymerase adds nucleotides extending the primers. Two duplex DNA copies are generated from the template.
2 mins 72 degC

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7
Q

How is the number of cycles related to the number of amplification?

A

DNA is amplified (2)^number of cycles

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8
Q

What is Taq I Polymerase and what is special about it?

A

a thermostable DNA polymerase, can be heated to high temperatures.

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9
Q

What is a thermocycler?

A

machine where PCR reaction is conducted.

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10
Q

What are some applications of PCR?

A

production of probes, gene cloning and manipulation, gene mutagenesis, DNA sequencing, amplification of ancient DNA, diagnostic in medicine, and forensic DNA typing.

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11
Q

What are non-coding sequences?

A

self replicated, selfish, no function expect their own reproduction. Ex: Alu elements

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12
Q

Where are Alu elements found?

A

Found in exons, but mostly exist in introns and other non-coding regions.

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13
Q

How are Alu elements replicated?

A

replication is through an RNA intermediate, copied into dsDNA called retrotransposon that inserts randomly elsewhere in the genome.

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14
Q

Polymorphisms

A

DNA sequence which varies between individuals.

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15
Q

Dimorphic

A

insertions that might be present or absent in each homologous chromosomes.

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16
Q

Is the Alu sequence dimorphic?

A

yes

17
Q

Where is the Alu sequence found in the chromosome?

A

found on a section of chromosome 16 PV92 locus.

18
Q

How many base pairs is chromosome 16 PV92 locus?

A

this section of DNA is 700 bp, insertion of Alu sequence (300 bp) increases the length of the region to 1000 bp.

19
Q

What does one band at 1000 bp indicate on agarose gel?

A

Individual is homozygous for Alu insertion

20
Q

What does one band at 1000 bp and second band at 700 bp indicate on agarose gel?

A

Individual is heterozygous for Alu insertion

21
Q

What does one band at 700 bp indicate on agarose gel?

A

Individual is homozygous for no Alu insertion

22
Q

What is gel electrophoresis of DNA?

A

used to monitor DNA cleavage at alkaline pH.

23
Q

What charge is DNA and RNA?

A

negatively charged.

24
Q

What size of DNA fragments is PAGE used for?

A

small DNA fragments.

25
Q

What size of DNA fragments is agarose gel used for?

A

check larger DNA molecules, separation depends on length of DNA molecule.