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B12 > DNA Manipulation (13.1) > Flashcards

DNA Manipulation (13.1) Flashcards

1
Q

State what the PCR technique is used for

A

DNA amplification

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2
Q

Describe gel electrophoresis

A

Method used to separate and visualise nucleic acids and proteins according to their size

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3
Q

State what process DNA amplification uses to create a large quantity of DNA

A

PCR

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4
Q

State what PCR stands for

A

Polymerase Chain Reaction

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5
Q

State what DNA amplification enables

A

The rapid and accurate replication of target DNA

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6
Q

Describe ‘target DNA’

A

Particular region of a DNA molecule intended to be manipulated/studied

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7
Q

State what PCR is based on

A

The action of polymerases

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8
Q

Describe polymerases

A

Enzymes that catalyse the formation of polymers in nucleic acids

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9
Q

State the 2 different different groups of polymerases

A
  1. DNA polymerase

2. RNA polymerase

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10
Q

State the function of DNA polymerase

A

Acts to assemble DNA

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11
Q

State what polymerases can be classified as

A

Enzymes

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12
Q

Provide 2 examples of target DNA

A
  1. Specific gene

2. Microsatellite

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13
Q

Describe microsatellite

A

Short repeated sequence of nucleotides found at a defined locus on a chromosome.

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14
Q

State what makes microsatellites useful for DNA profiling

A

Variation in the number of repeats between individuals

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15
Q

State what the DNA polymerase enzyme uses as a template for building a new DNA strand

A

Each strand of the DNA double helix

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16
Q

State what each double stranded DNA copy is identical to

A

The original DNA strand

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17
Q

State what enzyme is involved in the unzipping of double helix of DNA

A

Helicase enzymes

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18
Q

Describe the function of primers

A

Determine the start and end points of a nucleotide sequence to be amplified

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19
Q

State why primers are so critical for the function of DNA polymerase

A

DNA polymerase can only attach to the end of a nucleotide chain that is base paired with the template strand

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20
Q

Describe taq polymerase

A

Thermostable DNA polymerase that is most commonly used in PCR

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21
Q

State from where taq polymerase was sought

A

Thermophilic bacteria

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22
Q

Describe the function of RNA polymerase

A

Acts to assemble RNA

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23
Q

State what RNA polymerase synthesises in cells

A

mRNA, rRNA and tRNA

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24
Q

State how RNA polymerase synthesises RNA strands in cells

A

Transcription of genes

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25
Q

State whether or not RNA polymerase requires a primer to anneal to the DNA template strand to start synthesis

A

No.

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26
Q

State until when RNA polymerase adds nucleotides

A

STOP codon is reached

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27
Q

State whether RNA polymerase or DNA polymerase functions faster

A

RNA polymerase works much more slowly

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28
Q

Describe reverse transcriptase

A

DNA polymerase that synthesises single-stranded DNA using single-stranded RNA as a template

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29
Q

State what the function of reverse transcriptase is the reverse of

A

The usual transcription process in which DNA is transcribed into RNA

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30
Q

State what reverse transcriptase is used to produce

A

DNA molecules that can be amplified by PCR for further analysis

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31
Q

State what reverse transcriptase is also used to make and from what

A

Complementary DNA (cDNA) from mRNA

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32
Q

State whether or not complementary DNA produced by reverse transcriptase includes or does not include introns

A

Introns are sliced out. Therefore, does not include.

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33
Q

State what PCR is a method of

A

Amplifying specific target sequences of DNA

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34
Q

State whether or not taq polymerase is used in the PCR method

A

Yes.

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35
Q

State why taq polymerase is used in the PCR method

A

Resists changes in temperature required during PCR

36
Q

State what each cycle of PCR produces

A

2^n DNA

37
Q

State what a PCR mixture requires

A
  1. DNA
  2. Free nucleotides
  3. Heat resistant DNA polymerase
  4. DNA primers (x2)
38
Q

State whether or not target DNA to be amplified is also included in the PCR mixture

A

Yes.

39
Q

State why free nucleotides are included in the PCR mixture

A

Used to build new DNA strands

40
Q

State why heat-resistant DNA polymerase are included in the PCR mixture

A

Elongate the new DNA strands by adding the free nucleotides

41
Q

Provide an example of a the heat-resistant DNA polymerase commonly used throughout PCR

A

Taq polymerase

42
Q

State why DNA primers are complementary to the ends of the target DNA

A

Specify start and finish of the DNA sequence to be amplified

43
Q

State why DNA primers are created

A

To form hydrogen bonds with complementary base pairs on either end of DNA sequence to be amplified

44
Q

State where the PCR mixture is placed

A

DNA thermocycler

45
Q

State what DNA thermocycler does for the PCR mixture

A

Alters the temperature over three steps

46
Q

State the 3 major steps of PCR

A
  1. Denaturation
  2. Annealing
  3. Extension
47
Q

Describe denaturation as the first step in the DNA thermocycler

A

Sample heated to 95C

48
Q

State why the sample is first heated to 95C when placed in the DNA thermocylcer

A

To break hydrogen bonds between the two strands of double-stranded DNA to obtain single strands

49
Q

Describe annealing as the second step in the DNA thermocycler

A

Temperature is reduced to 50-60C

50
Q

State why the temperature of the DNA thermocycler is decreased to 50-60C in the second stage

A

Allows primers to bind to complementary sequences on opposite strands at end of target DNA sequence

51
Q

Describe extension as the third step in the DNA thermocycler

A

Temperature is increased to 72C

52
Q

State why the temperature of the DNA thermocycler is increased to 72C

A

Allows taq polymerase to attach to the primers on the DNA strands

53
Q

State approximately how many times the heating and cooling cycle is repeated in the DNA thermocycler

A

Up to 50 times

54
Q

State what the second stage of the DNA thermocycler process is known as

A

Annealing

55
Q

State what the first stage of the DNA thermocycler process is known as

A

Denaturation

56
Q

State what the third stage of the DNA thermocycler process is known as

A

Extension

57
Q

State what gel electrophoresis can be used to do (in addition to separating DNA and RNA)

A

Separate proteins of different sizes

58
Q

Describe when gel electrophoresis is commonly performed

A

After DNA amplification

59
Q

State what gel electrophoresis allows scientists to do

A

Separate DNA fragments in a sample based on size

60
Q

Describe gel electrophoresis

A

Technique used for separating fragments of nucleic acids

61
Q

State why DNA is negatively charged

A

Negative charge on the phosphate group in each nucleotide

62
Q

State what occurs to negatively charged DNA molecules when an electric current is applied to the electrophoresis gel

A

Negatively charged DNA molecules move towards positive terminal

63
Q

State 3 reasons for which gel electrophoresis is used to compare DNA fragments

A
  1. DNA screening
  2. Confirming the correct gene has been amplified in PCR
  3. Identifying DNA fragments obtained by restriction enzyme digestion
64
Q

State what gel involved in electrophoresis is composed of

A

Agarose

65
Q

Describe agarose

A

Purified form of agar

66
Q

State at what terminal of the chamber the wells are situated

A

Negative terminal

67
Q

State where each DNA sample is loaded into

A

Within the gel of one of the wells

68
Q

Describe DNA ladder

A

Set of DNA molecules of known size used to determine the size of other molecules

69
Q

State what can be used in the gel electrophoresis process to estimate the length of the sample DNA fragments

A

DNA ladder

70
Q

State what the gel placed in an electrophoresis bath is covered in

A

Controlled pH solution

71
Q

State what the controlled pH solution introduced to the gel placed in an electrophoresis bath contains

A

Ions to conduct an electric current

72
Q

State what the electrical current introduced to the gel electrophoresis bath causes

A

Negatively charged DNA fragments to migrate towards the positive terminal of the chamber

73
Q

State what fragments move fastest in the gel when an electrical current is applied

A

Smaller fragments

74
Q

State how DNA fragments are detected

A

Stain application that binds to DNA

75
Q

State 2 examples of stain applied to detect DNA fragments

A
  1. Methyl blue

2. Fluorescent

76
Q

State what the smearing of bands in the gel indicates

A

Overloading of wells/degradation of the DNA or protein

77
Q

State the 3 ways PCR and electrophoresis can be used to detect a mutant allele

A
  1. Isolate individual’s DNA
  2. Use PCR primers complementary to DNA sequences
  3. Compare amplified DNA molecules
78
Q

State why PCR primers that are complementary to the DNA sequences on either side of the site of the mutation are used to detect a mutant allele

A

Amplify DNA

79
Q

Describe well

A

An indent in the gel into which a DNA sample is loaded

80
Q

Describe standard ladder

A

A mixture of DNA fragments of known length that are used in order to infer the size of the fragments in the sample

81
Q

Describe agarose gel

A

A sponge-like gel used in gel electrophoresis that contains pores for DNA fragments to move through

82
Q

Describe buffer

A

An ion-filled solution that carries current though agarose gel

83
Q

Describe electrode

A

Conductors of electricity that enable current to pass through gel

84
Q

Describe band

A

A line seen in the gel after running gel electrophoresis that corresponds to a collection of DNA fragments of a specific size

85
Q

Describe ethidium bromide

A

A fluorescent dye that binds to DNA fragments in a gel and allows them to be visualised

86
Q

Describe recognition site

A

A specific target sequence of DNA upon which a restriction enzyme acts

B12 (41 decks)

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