Lab 6.

Question 1

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

Answer 1

To collect a sample human tissue from the crime

scene or victim.

Question 2

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

Answer 2

Blood.

Hair.

Skin.

Body fluids.

Question 3

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

Answer 3

As a stain.

Question 4

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

Answer 4

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

Question 5

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

Answer 5

The polymorphic regions of the sample are amplified by PCR.

Question 6

What are polymorphic regions of DNA?

Answer 6

Regions that vary in length depending on the individual.

Question 7

What are the 2 categories of polymorphic regions within DNA?

Answer 7

Variable Number of Tandem Repeats (VNTR).

Short Tandem Repeats (STR).

Question 8

What is a VNTR?

Answer 8

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

Question 9

What is an STR?

Answer 9

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

2-4 nucleotides in length.

Question 10

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

Answer 10

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

Question 11

Where cant the VNTR D1S80 be found?

Answer 11

On chromosome 1.

Question 12

What sequence doe the VNTR D1S80 contain?

Answer 12

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

Question 13

What will an individual who is homozygous for the

D1S80 genotype have on chromosome 1?

Answer 13

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

Question 14

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

Answer 14

There will be a single PCR product following gel analysis.

Question 15

What will an individual who is heterozygous for the

D1S80 genotype have on chromosome 1.

Answer 15

They will have differing D1S80 repeat numbers.

Question 16

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

Answer 16

There will be 2 different PCR products.

Question 17

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

Answer 17

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

Question 18

What enzyme is used in PCR?

Answer 18

Taq polymerase.

Question 19

Where is Taq polymerase obtained from?

Answer 19

From bacteria that are found in hot springs.

Question 20

Why is TAQ polymerase used in PCR?

Answer 20

Because it is stable at very high temperatures.

Question 21

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

Answer 21

Primers.

Question 22

How long are the oligonucleotides that are used in PCR?

Answer 22

15-30 nucleotides.

Question 23

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

Answer 23

The template.

Question 24

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

Answer 24

The target.

Question 25

What happens in the first step of PCR?

Answer 25

The complementary DNA strands of the template are separated.

Question 26

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

Answer 26

They are denatured at 94 degrees C.

Question 27

What happens in the 2nd step of PCR?

Answer 27

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

Question 28

What happens in the 3rd step of PCR?

Answer 28

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

Question 29

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

Answer 29

Denaturation. Annealing. Extension.

Question 30

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

Answer 30

Around 30-40 cycles.

Question 31

What was the objective of lab 6?

Answer 31

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

Question 32

What cells were analysed via PCR?

Answer 32

Hair or cheek cells.

Question 33

What locus was the DNA amplified at during lab 6?

Answer 33

At the D1S80 locus by PCR.

Question 34

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

Answer 34

To ensure that there is enough DNA in the sample.

Question 35

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

Answer 35

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

Question 36

What does polymorphic DNA refer to?

Answer 36

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

Question 37

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

Answer 37

The examination of polymorphic regions within an individuals genome.

Question 38

What is a VNTR?

Answer 38

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

Question 39

What kind of individuals will share a DNA fingerprint?

Answer 39

Identical twins.

Question 40

Who started DNA fingerprinting?

Answer 40

Dr. Alex Jeffreys at the University of Leicester.

Question 41

What is the Combined DNA Index System (CODIS)?

Answer 41

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

Question 42

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

Answer 42

A convicted offender index. A forensic index.

Question 43

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

Answer 43

It indicates a suspect.

Question 44

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

Answer 44

It indicates a serial offender.

Question 45

What 4 things can DNA polymorphisms be used to detect?

Answer 45

Paternity and maternity. Kinship. The identification of human remains. For criminal forensics.

Question 46

PCR is performed in what kind of machine?

Answer 46

A thermal cycler.

Question 47

What is a thermal cycler?

Answer 47

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

Question 48

What solution was used to extract cheek cells?

Answer 48

A saline solution.

Question 49

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

Answer 49

Around 1-2 hours.

Question 50

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

Answer 50

5 μL of gel loading dye.

Question 51

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

Answer 51

The samples are placed in the freezer.

Question 52

What happens to the amplified DNA before electrophoresis?

Answer 52

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

Question 53

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

Answer 53

29 different alleles.

Question 54

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

Answer 54

The length of the alleles within the DNA sample.

Question 55

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

Answer 55

The molecular weight standard DNA fragments.

Question 56

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

Answer 56

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

Question 57

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

Answer 57

A 10% margin of error.

Question 58

How are the sizes of the PCR products extrapolated?

Answer 58

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

Question 59

How is a primer dimer represented on the electrophoresis gel?

Answer 59

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

Question 60

What is a primer dimer?

Answer 60

Primers that have amplified themselves after PCR.

Question 61

What happens if no band is visible after electrophoresis?

Answer 61

It usually occurs due to experimental error.

Question 62

What happens if 1 band is visible after electrophoresis?

Answer 62

Either, the person is homozygous at the D1S80 locus. Or theother band failed to amplify.

Question 63

What happens if 2 bands are visible after electrophoresis?

Answer 63

The person is heterozygous at the D1S80 allele.

Question 64

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

Answer 64

The primers may randomly bind to other chromosomal loci.

Question 65

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

Answer 65

The two brightest bands are usually the true alleles.

Question 66

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

Answer 66

By changing the annealing temperature of the PCR reaction.

Question 67

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

Answer 67

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

Question 68

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

Answer 68

It removes Mg.

Question 69

What is required to obtain cell lysis?

Answer 69

Boiling.

Question 70

Does boiling degrade DNA?

Answer 70

No.

Question 71

Why will nucleases not degrade the DNA?

Answer 71

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

Question 72

What does the PCR reaction pellet contain?

Answer 72

Taq polymerase. The 4 dNTPs. Mg2+. The buffer.