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Molecular Biology Lab. > Lab 6. > Flashcards

Lab 6. Flashcards

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1
Q

What is the first step in DNA fingerprinting at a crime scene?

A

To collect a sample human tissue from the crime

scene or victim.

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2
Q

What tissues from a crime scene can be used for DNA fingerprinting?

A

Blood.

Hair.

Skin.

Body fluids.

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3
Q

What is the sample of human tissue that is found at a crime scene often present as?

A

As a stain.

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4
Q

How is the sample of human tissue at a crime scene treated?

A

With a detergent that will lyse cell membranes allowing access to the DNA.

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5
Q

What happens to the genetic sample that is obtained from a crime scene after it has been obtained?

A

The polymorphic regions of the sample are amplified by PCR.

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6
Q

What are polymorphic regions of DNA?

A

Regions that vary in length depending on the individual.

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7
Q

What are the 2 categories of polymorphic regions within DNA?

A

Variable Number of Tandem Repeats (VNTR).

Short Tandem Repeats (STR).

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8
Q

What is a VNTR?

A

A region of DNA that is composed of a 15-70 BP sequence that is repeated around 5-100 times.

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9
Q

What is an STR?

A

It is similar to a VNTR, but the repeated section is only

2-4 nucleotides in length.

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10
Q

How can investigators obtain a unique DNA fingerprint for an individual?

A

By looking at the VNTRs or STRs from the same individual.

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11
Q

Where cant the VNTR D1S80 be found?

A

On chromosome 1.

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12
Q

What sequence doe the VNTR D1S80 contain?

A

A 16 nucleotide sequence that is variably repeated between 16 and 40 times.

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13
Q

What will an individual who is homozygous for the

D1S80 genotype have on chromosome 1?

A

They will have an equal number of repeats on both homologues of chromosome 1.

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14
Q

How many products are displayed after PCR electrophoresis for the DS180 if it comes from an individual who is homozygous for the D1S80 genotype?

A

There will be a single PCR product following gel analysis.

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15
Q

What will an individual who is heterozygous for the

D1S80 genotype have on chromosome 1.

A

They will have differing D1S80 repeat numbers.

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16
Q

How many products are displayed after PCR electrophoresis for the DS180 if it comes from an individual who is heterozygous for the D1S80 genotype?

A

There will be 2 different PCR products.

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17
Q

Why do law enforcement agencies prefer to use STRs over VNTRs?

A

As they are more easily amplified and require less DNA for analysis.

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18
Q

What enzyme is used in PCR?

A

Taq polymerase.

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19
Q

Where is Taq polymerase obtained from?

A

From bacteria that are found in hot springs.

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20
Q

Why is TAQ polymerase used in PCR?

A

Because it is stable at very high temperatures.

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21
Q

What are the oligonucleotides that are used in PCR known as?

A

Primers.

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22
Q

How long are the oligonucleotides that are used in PCR?

A

15-30 nucleotides.

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23
Q

What is the extracted region of DNA that is used in PCR known as?

A

The template.

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24
Q

What is the region of the template DNA that is to be amplified via PCR known as?

A

The target.

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25
Q

What happens in the first step of PCR?

A

The complementary DNA strands of the template are separated.

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26
Q

How are the DNA strands of the template separated during PCR?

A

They are denatured at 94 degrees C.

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27
Q

What happens in the 2nd step of PCR?

A

The sample is cooled to around 40-65 degrees C so that 1 of the 2 DNA strands can hybridise to the 2 primers.

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28
Q

What happens in the 3rd step of PCR?

A

The temperature is raised to 72 degrees C and Taq P adds nucleotides to the primers to create a new strand.

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29
Q

What are the 3 steps of PCR that occur in a single cycle of PCR?

A

Denaturation.

Annealing.

Extension.

30
Q

How many cycles of PCR are usually done during a typical experiment?

A

Around 30-40 cycles.

31
Q

What was the objective of lab 6?

A

To isolate DNA and compare the polymorphisms between samples by PCR amplification and gel electrophoresis.

32
Q

What cells were analysed via PCR?

A

Hair or cheek cells.

33
Q

What locus was the DNA amplified at during lab 6?

A

At the D1S80 locus by PCR.

34
Q

Why is it important to ensure that there are enough cells in the sample that is extracted from the DNA donor?

A

To ensure that there is enough DNA in the sample.

35
Q

How should used disposbale equipment be treated before it is thrown away?

A

It should be placed into a 15% bleach solution for 15 minutes and then thrown away.

36
Q

What does polymorphic DNA refer to?

A

To the varibale chromosomal regions that are found in different individuals.

37
Q

What factors about DNA can lead to the creation of a DNA fingerprint?

A

The examination of polymorphic regions within an individuals genome.

38
Q

What is a VNTR?

A

A region of DNA that has a 15-70 BP sequence that is tandemly arranged from heal to tail.

39
Q

What kind of individuals will share a DNA fingerprint?

A

Identical twins.

40
Q

Who started DNA fingerprinting?

A

Dr. Alex Jeffreys at the University of Leicester.

41
Q

What is the Combined DNA Index System (CODIS)?

A

A system that compares crime scene DNA to DNA profiles stored in 2 different databases.

42
Q

What are the 2 databses in CODIA that will be used to compare crime scene DNA to known DNA?

A

A convicted offender index.

A forensic index.

43
Q

What happens if crime scene DNA matches to a profile in the convicted offender index?

A

It indicates a suspect.

44
Q

What happens if crime scene DNA matches to a profile in the forensic index?

A

It indicates a serial offender.

45
Q

What 4 things can DNA polymorphisms be used to detect?

A

Paternity and maternity.

Kinship.

The identification of human remains.

For criminal forensics.

46
Q

PCR is performed in what kind of machine?

A

A thermal cycler.

47
Q

What is a thermal cycler?

A

An instrument that will rapidly heat and cool samples to different temperatures.

48
Q

What solution was used to extract cheek cells?

A

A saline solution.

49
Q

How long does the PCR progrma run for while performing this experiment?

A

Around 1-2 hours.

50
Q

What is added to each sample after PCR has been completed?

A

5 μL of gel loading dye.

51
Q

What happens to the PCR samples after the gel loading dye has been added to the sample?

A

The samples are placed in the freezer.

52
Q

What happens to the amplified DNA before electrophoresis?

A

A 200 BP DNA ladder is heated in a water bath at 50°C for 2 minutes.

53
Q

How many different alleles have been found at the D1S80 locus by population studies?

A

29 different alleles.

54
Q

What could we determine from the electrophoresis results after lab 6?

A

The length of the alleles within the DNA sample.

55
Q

What factor about the electrophoresis helped us to identify the size of the alleles?

A

The molecular weight standard DNA fragments.

56
Q

What is the first step of determining the size of the D1S80 alleles?

A

To determine the migration distance and size of the DNA PCR products after electrophoresis.

57
Q

What margin of error can be used when estimating the size of the DNA samples or alleles?

A

A 10% margin of error.

58
Q

How are the sizes of the PCR products extrapolated?

A

By drawing a graph that shows their migration distance relative to the Standard DNA fragments.

59
Q

How is a primer dimer represented on the electrophoresis gel?

A

As a fuzzy band that appears at the same location in each lane which will be near the bottom of the gel.

60
Q

What is a primer dimer?

A

Primers that have amplified themselves after PCR.

61
Q

What happens if no band is visible after electrophoresis?

A

It usually occurs due to experimental error.

62
Q

What happens if 1 band is visible after electrophoresis?

A

Either, the person is homozygous at the D1S80 locus.

Or theother band failed to amplify.

63
Q

What happens if 2 bands are visible after electrophoresis?

A

The person is heterozygous at the D1S80 allele.

64
Q

What happens if 3 or more bands are visible after electrophoresis?

A

The primers may randomly bind to other chromosomal loci.

65
Q

What bands represent the true alleles in an electrophoresis sample that contains 3 or more bands?

A

The two brightest bands are usually the true alleles.

66
Q

How can an experiment that gets 3 or more bands be imporved?

A

By changing the annealing temperature of the PCR reaction.

67
Q

What is found in the lysis solution that was used in this experiment?

A

25 mM Tris-HCl, pH 8.0 and 5% chelating agent.

68
Q

What is the function of the chelating agent that is found in the lysis solution?

A

It removes Mg.

69
Q

What is required to obtain cell lysis?

A

Boiling.

70
Q

Does boiling degrade DNA?

A

No.

71
Q

Why will nucleases not degrade the DNA?

A

The need magnesium for a co-factor and it has been removed.

72
Q

What does the PCR reaction pellet contain?

A

Taq polymerase.

The 4 dNTPs.

Mg2+.

The buffer.

Molecular Biology Lab. (7 decks)

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