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Molecular Biology. > Polymerase Chain Reaction and Southern Blot. > Flashcards

Polymerase Chain Reaction and Southern Blot. Flashcards

1
Q

Define a polymorphism?

A

When more than 1 allele occupies a genes locus.

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2
Q

Define a point mutation?

A

A mutation that only affects a single nucleotide.

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3
Q

Define a restriction fragment length polymorphism

A

It is the variation of length in a DNA fragment that is produced by a specific restriction enzyme

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4
Q

Define a VNTR?

A

A variable nucleotide tandem repeat.

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5
Q

Where are VNTR’s most likely to be found?

A

They are found all over the chromosome but are most often found near the centromere.

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6
Q

What does the combination of an RFLP and Southern blot analysis allow scientists to identify?

A

Insertions and deletions within a gene.

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7
Q

What does Southern blot analysis allow us to identify?

A

A specific gene in a genome when the genes have been separated by electrophoresis and transferred to a Southern blot membrane.

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8
Q

How is the specific DNA sequence on a Southern blot membrane recognised?

A

A probe in the Southern blot will recognise a matching sequence within the DNA.

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9
Q

What does Southern blot analysis help to locate within a persons genome?

A

To locate insertions and deletions within a persons genome.

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10
Q

What are the 4 steps of Southern blot?

A

The DNA is digested by restriction endonuclease’s and the fragments are sorted by gel electrophoresis.

DNA is transferred to a Southern blot membrane which is made of nitrocellulose.

A specific probe is then added to the membrane and this probe it hybridises to a specific DNA fragment.

The membrane is analysed via X-ray and it will show the radioactive probe bound to the gene of interest.

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11
Q

How are the DNA fragments from a Southern blot compared with each other?

A

From the position of each specific gene on the gel that is used for electrophoresis.

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12
Q

Where do the genes that are compared via Southern blot come from?

A

From different people, there is also a control gene that represents the standard gene.

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13
Q

How will DNA strands that have suffered from deletions or insertions differ from each other?

A

DNA that has had deletions will move further up the gel than the control as it will be lighter and will move faster.

DNA that has had insertions will be located below the control as it will be heavier and more slowly.

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14
Q

What are VNTR’s?

A

They are repeating sequences of DNA that often lie close to the centromere on a chromosome.

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15
Q

What part of a genome is used for Southern blot in a paternity test?

A

VNTR’s.

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16
Q

Why are VNTR’s used for performing a paternity test via Southern blot?

A

As each person contains a different number of base’s within their VNTR’s.

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17
Q

What is analysed in the VNTR’s when performing a paternity test via Southern blot?

A

The number of base’s within the VNTR’s.

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18
Q

Why are VNTRs used when performing a paternity test via Southern blot?

A

As one chromosome comes from the mother and the other comes from the father.

Therefore, one of the babys VNTR’s should be the same on the Dad the other should be the same on the Mum.

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19
Q

How is a paternity test via Southern blot carried out?

A

DNA is isolated from the suspected father, the mother and the child and compared via Southern blotting.

It is spearated by electrophoresis and then placed on the filter.

Once on the Southern blot filter, the probes can be compared.

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20
Q

How will the DNA samples look if the suspected father is the father of the baby in a paternity test via Southern blot?

A

The electrophoresis picture will contain 2 bands for each person.

If the baby belongs to the father then 1 band should be identical to 1 of the fathers bands.

The other band should be identical to 1 of the mothers band.

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21
Q

Can point mutations destroy restriction sites within DNA?

A

Yes.

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22
Q

How can the absence of restriction sites be tested for?

A

Via Southern blot.

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23
Q

What is the point mutation that causes sickle cell anaemia?

A

The change of a single nucleotide that codes for valine instead of glutamic acid.

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24
Q

Does the point mutation that leads to sickle cell destroy a restriction site?

A

Yes.

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25
Q

Can sickle cell be proven via Southern blot analysis?

A

Yes.

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26
Q

What genetic samples are removed used to test for sickle cells disease?

A

The samples are cleaved at a restriction site that is upstream of the restriction site of interest.

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27
Q

The genetic sample that is used to test for sickle cell is removed from the DNA strand via the use of what enzyme?

A

MST-2.

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28
Q

How will the genetic samples of person who has scikle cell compare with the sampe of a person who does not have sickle cell when analysed via Southern blot?

A

A person who does not have sickle cell has a sample that is higher up the electrophoresis graph as it contains fewer base pairs.

A person who has sickle cell will have a sample that is lower down the electrophoresis graph as it contains more DNA.

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29
Q

How many base pairs are usually found in the Southern blot sample of a person who does not have sickle cell?

A

Around 1150 base pairs

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30
Q

How many base pairs are usually found in the Southern blot sample of a person who has sickle cell?

A

Around 1350 base pairs

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31
Q

Why is the Southern blot sample of a person who does not have sickle cell have fewer base pairs?

A

Because their genome contains the complete restriction site, meaning that the sample is cleaved at that site.

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32
Q

Why is the Southern blot sample of a person who has sickle cell have more base pairs?

A

As the genome does not have the restriction site, meaning that the sample is cleaved at the next site so it contains more BPs.

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33
Q

Where are the cleavage sites on a Southern blot sample from a person that has sickle cell?

A

At the upstream site.

At the site that is downstream from where the gene of interest should be.

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34
Q

How many sequences will a Southern blot sample for a person who is heterozygous for sickle cell contain?

A

There are 2 sequences.

One chromosome will contain the short sequence and the restriction site of interest.

The other with have the heavy sequence and no restriction site.

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35
Q

Why can a person who does not have sickle cell have 2 possible sequences on a Southern blot sample?

A

As the probe can bind upstream or downstream of the restriction site of interest.

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36
Q

What are the 2 possible amounts of base pairs that can be found on a Southern blot sample of a person who does not have sickle cell?

A

If the probe binds upstream of the site of interest it will be 1150 BPs.

If the probe binds downstream of the site of interest then there will be 200 base pairs.

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37
Q

Who invented the PCR reaction?

A

Kary Mullis in 1983.

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38
Q

What is the PCR reaction used for?

A

For the rapid production of large amounts of specific DNA sequences.

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39
Q

Is the process of PCR automatic?

A

Yes.

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40
Q

How does the amount of DNA increase after each round of PCR?

A

It doubles the amount of DNA.

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41
Q

What are the 6 components required for successful PCR?

A

DNA polymerase.

Primers.

DNTPs.

Magnesium chloride.

A buffer.

DNA.

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42
Q

What is the DNA polymerase used for in PCR?

A

To synthesise a new strand of DNA that is complimentary to an existing single strand of DNA.

43
Q

What is the primer used for in PCR?

A

Their presence on the DNA strand will help DNA polymerase attach to the strand.

44
Q

What are the DNTPs that used for in PCR?

A

These are de-oxynucleotide triphosphate’s and they differ in 4 ways depending on their base pair.

45
Q

What are the 4 DNTPs used in PCR?

A

DATP.

DGTP.

DCTP.

DTTP.

46
Q

What is magnesium chloride used for in PCR?

A

As a co-factor for DNA polymerase.

47
Q

What is the buffer used for in PCR?

A

To ensure that reaction conditions remain stable.

48
Q

What is the DNA used for in PCR?

A

As a complimentary template for the sequence of new DNA.

49
Q

What are the 4 steps of PCR?

A

Denaturation.

Annealing.

Primer extension.

Repetition.

50
Q

What occurs in the denaturation step of PCR?

A

The target DNA is heated to 95 degrees and denatured.

51
Q

What happens when the DNA is denatured during the 1st step of PCR?

A

The hydrogen bonds are broken and the 2 strands separate to form 2 single strands.

52
Q

What happens in the annealing stage of PCR?

A

The DNA is cooled to between 45 and 65 degrees.

This allows the RNA primers to bind to a complimentary strand.

53
Q

The optimum temperature for the binding of the primers to the DNA during PCR depends on what?

A

The properties of the primer.

54
Q

What are the names of the 2 primers invovled in PCR?

A

The forward and reverse primers.

55
Q

How does the forward primer read the DNA strand?

A

From left to right.

56
Q

How does the reverse primer read the DNA strand?

A

From right to left.

57
Q

What areas of the DNA strand will the primers bind to during PCR?

A

The primers bind to flanking sequences that are on each side of the target sequence.

58
Q

What happens during primer extension in PCR?

A

DNA polymerase II use’s MgCl as a co-factor.

The polymerase will extend the primer in the 5 to 3 direction and it will read the strand in the 3 to 5 direction.

59
Q

What type of polymerase is used in PCR?

A

A TAQ polymerase as it is capable of withstanding all of the temperatures in this process.

60
Q

What is the optimum temperature of a TAQ polymerase?

A

72 degrees.

61
Q

How many DNA molecules are formed at the end of the primer extension phase of PCR?

A

2 molecules of double stranded DNA.

62
Q

What happens during the repetition phase of PCR?

A

The previous steps are repeated as many times as necessary.

63
Q

What happens to the amount of DNA molecules after every cycle of PCR?

A

The number of DNA molecules doubles.

64
Q

What is the TAQ polymerase isolated from?

A

A bacteria that lives in hot springs and hydrothermal vents.

65
Q

What part of the PCR reaction allows for automation and why?

A

The TAQ polymerase as other polymerase’s denature at 95 degrees meaning that they have to be replaced.

66
Q

What is the half life of a TAQ polymerase?

A

Around 40 minutes at 95 degrees.

67
Q

What does TAQ polymerase add to a newly synthesised DNA strand?

A

An overhang that consists of a single adenine residue.

68
Q

When can the overhang added to a DNA strand by TAQ polymerase be exploited?

A

When the fragment is being cloned into vectors as it serves as a natural sticky end.

69
Q

Which TAQ polymerase has proof reading ability?

A

Hi-fidelity TAQ

70
Q

What are the 3 phases that are involved in the overall PCR reaction?

A

Exponential phase.

Linear phase.

Endpoint.

71
Q

What occurs during the exponential phase of PCR?

A

The product is doubling at every cycle.

72
Q

What occurs during the linear phase of PCR?

A

The reaction substrates are slowly consumed and accumulation of the product slows.

73
Q

What occurs during the endpoint phase of PCR?

A

Substrates are exhausted and no more product accumulates.

74
Q

The substrates used in PCR usually last for how many cycles?

A

Around 30 cycles.

75
Q

What is a good primer that can be used for PCR?

A

It must be long enough to be specific, but short enough to easily bind to the target sequence.

76
Q

What should the melting point of a PCR primer be?

A

Between 52 and 58 degrees.

77
Q

What should the GC content of a PCR primer be?

A

Between 40-60%. as this ensures that the melting temperature is correct.

78
Q

What are 5 applications of PCR?

A

Re-sequence a gene from any DNA sample.

Test blood for the presence of a virus.

Embryo genotyping.

Surveillance of mutated cancer cells.

Forensic sciences.

79
Q

What are 4 molecular applications of PCR?

A

PCR based mutagenesis.

Cloning strategies.

Gene expression analysis.

Genomics.

80
Q

Where must the primers for PCR be designed and why is this a drawback?

A

They must be designed in a lab.

This there must be knowledge about the sequence information on the target DNA.

81
Q

Is PCR easy to contaminate?

A

Yes, as a result it needs strict controls.

82
Q

When is it difficult to amplify DNA in PCR?

A

When the DNA is longer than 1500 BP.

83
Q

Why does PCR have limitations when involved in electrophoresis?

A

As PCT has low sensitivity, meaning that a certain amount of DNA must be used at the beginning if it is to show up after electrophoresis.

84
Q

What is the major drawback of using PCR and electrophoresis?

A

Electrophoresis only gives size based discrimination, so the results are not expressed quantitatively.

85
Q

What is real time PCR based around?

A

The detection and quantification of a fluorescent reporter that increase’s every time the sample doubles in size.

86
Q

What is the baseline in real time PCR?

A

The baseline consists of all of the components in the reaction except for the template.

87
Q

What is the threshold in real time PCR?

A

It is ten times the rate of the baseline.

88
Q

What is CT in real time PCR?

A

CT is the cycle number which is when the fluorescence crosse’s the threshold.

89
Q

What is the plateau determined by in real time PCR?

A

The amount of primers in the experiment.

90
Q

Does real time PCR require gel based analysis?

A

No, as analysis is based around the computer detection of fluorescence.

91
Q

What are the 2 major adavantages of using real time PCR?

A

Amplification can be monitored in real time.

It does not have low sensitivity so it can begin with small amounts of DNA as a template.

92
Q

What are the 3 types of probes used in PCR?

A

Hydrolysis probes.

Hybridisation or FRET probes.

DNA binding agents.

93
Q

What kind of PCR probe is the TAC man probe?

A

A hydrolysis probe.

94
Q

What 2 molecules does the PCR TAC man probe contain?

A

A 5-reporter molecule and a 3-quencher molecule.

95
Q

How does the PCR TAC man probe work?

A

The probe binds to a PCR product and degrades.

The degradation of the probe separates the quencher and reporter allowing fluorescent emissions to be detected.

96
Q

How does the BEACON fluoresent probe work during PCR?

A

The beacon probe forms a hairpin loop structure when it is not bound to the DNA of interest.

When it comes into contact with the DNA of interest, the loop structure will be lost and it will bind to the DNA.

97
Q

What happens if the BEACON probe is not fully complimentary to the gene of interest?

A

It will not lose its hairpin loop and no bonds will be formed.

98
Q

How does the FRET fluoresent probe work during PCR?

A

The probe consists of a pair of primers that hybridise close to each other on the target DNA.

The upstream primer is labelled at the 3 prime end with a donor fluorescent molecule and the downstream primer is labelled at the 5 prime end with an acceptor fluorescent molecule.

The donor fluorescent molecule is excited by an external light source and it will pass on its excretory energy to the acceptor molecule which will emit light at a different wavelength which can be detected and measured.

99
Q

How does the SYBR green fluoresent probe work during PCR?

A

This probe binds to double stranded DNA and emits light after excitation.

100
Q

What is the amount of fluoresence in a SYBR green probe proportional to?

A

The amount of fluorescence emitted is directly proportional to the amount of product that is generated in the PCR reaction.

101
Q

When should the SYBR green fluoresent probe not be used for PCR?

A

if the PCR has not been optimised.

It should not be used to detect low level transcripts.

It should never be used in multiplex reactions.

102
Q

Is PCR or Soiuthern blot better at diagnosing sickle cell?

A

PCR.

103
Q

How is PCR used to diagnose sickle cell?

A

The gene of interest is amplified to create many copies.

The specific fragment is removed and placed on a gel.

The amount of base pairs is analysed to determine whether a person has sickle cell.

104
Q

What enzyme is used to remove the specific DNA fragment when diagnosing sickle cell via PCR?

A

dDE-1.

Molecular Biology. (22 decks)

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