DNA Fingerprinting/Profiling

Question 1

What is DNA fingerprinting/profiling?

Answer 1

A technique of determining individual’s characteristics which will be as unique as a fingerprint.

Question 2

Give some applications of these techniques?

Answer 2

Microbiology
Zoology
Forensic Science
Paternity testing
Immigration disputes

Question 3

What are the repetitive sequence of DNA (that are non-coding regions) known as?

Answer 3

Tandem Repeats and Dispersed Repeats

Question 4

What are the 3 categories of tandem repeats?

Answer 4

Satellites
Microsatellites
Minisatellites

Question 5

What is high copy number DNA?

Answer 5

They are highly repetitive short DNA sequences
AKA Satellite DNA
Composed of unequal ratios of AT/CG
Composed of long tandem repeats in heterochromatin

Question 6

What repetitive sequences with high copy numbers are found in the centromere of the dividing cell?

Answer 6

Alpha satellite DNA and Beta satellite DNA (both vary in length of bps)

Question 7

What is the structural function of repetitive DNA in telomeres?

Answer 7

Stop the chromosomes from sticking together and maintains the integrity of the chromosome while dividing.

Question 8

Why do we have repetitive DNA if they don’t make any contribution to phenotype (they are non-coding)?

Answer 8

Because they have just increased in number and abundance over evolutionary time.

Question 9

Where are repetitive sequences/satellite DNA found in the genome?

Answer 9

In the telomeres, centromeres and throughout the the chromosomal DNA.

Question 10

Describe how alpha satellite DNA is arranged?

Answer 10

Can have 171 bp monomeric repeats that go in either 3’-5’ or 5’-3’
or
All these different monomeric units can come together in a high order repeat (1-3kb) and these repeats can repeat.

Question 11

What are the sub-classes of Satellite DNA?

Answer 11

Mini and Micro satellite DNA

Question 12

How long is microsatellite DNA and where can it be found in the genome?

Answer 12

Typically less than 100 bp long including short tandem repeats (STR) and simple sequence repeats (SSR)
Found throughout the genome

Question 13

What is microsatellite DNA used for?

Answer 13

Modern DNA profiling

Question 14

How long is minisatellite DNA and where can it be found in the genome?

Answer 14

Typically 0.1-20kb LONG

Found near telomeres

Question 15

What is minisatellite DNA used for?

Answer 15

DNA fingerprinting

Question 16

Explain the principle/idea behind DNA fingerprinting

Answer 16

99.9% of DNA in all humans is the same but a small amount of it is different and unique. These unique minisatellite sequences are used to create a unique fingerprint of individuals and that is used when we need to identify people in various applications

Question 17

What are the two polymorphisms of satellite DNA?

Answer 17

1) Variable Number of Tandem Repeats (VNTRs)
(same unit repeated head to tail different number of times in different alleles)
2) Variable Structure DNA Repeats
(repeated many times in different alleles but can be next to other repeated sequences so not head to tail with itself)

Question 18

What are VNTRs?

Answer 18

Short nucleotide sequences (10-100bp) repeating in clusters

Each variant of this acts as an inherited allele

Question 19

How do we use VNTRs to distinguish between individuals?

Answer 19

The sequence is the same in all individuals but the number of repeats is different and that is the distinguishing part.
Band size on electrophoresis gel correlates to the number of times repeated (thicker band=more VNTRs)

Question 20

How was DNA fingerprinting first discovered and used?

Answer 20

Dr.Jeffreys was studying myoglobin and accidently observed varying DNA of individuals using restriction fragment length polymorphism.

Question 21

What does high stringency mean in terms of southern hybridisation analysis?

Answer 21

The conditions you use in order to get more specific binding of the probe to the DNA sequence.

Question 22

How does Restriction Fragment Length Polymorphism (RFLP) analysis work?

Answer 22

1) Use a DNA sample from a swab/saliva/blood
2) Cut DNA using restriction enzymes into small pieces
3) Separate the DNA fragments based on the size
4) Analyse using radiolabelled DNA repeat sequence

Question 23

What are the steps of Southern Hybridisation/DNA Blotting?

Answer 23

1) Extract the genomic DNA
2) Digest with RE
3) Fractionate by agarose gel electrophoresis
4) Blot onto a membrane
5) Hybridise with a radiolabelled myoglobin minisatellite sequence probe under low stringency conditions so the probe will bind to target DNA and related sequences
6) The higher up the probe binds in the gel, the more times the sequence is repeated here.

Question 24

When is the banding pattern different in a DNA fingerprint?

Answer 24

Different in different individuals so different sources of DNA used

Question 25

How can we increase the likelihood that an individual's sequence is unique to them?

Answer 25

Look at more than one repeating sequence on different loci in the genome

Question 26

What are the 2 variants of the 16 bp core of myoglobin probe? Hint: they are multilocus probes

Answer 26

33.6 and 33.15

Question 27

What is the probability that two individuals would share a fingerprinting band by chance?

Answer 27

0.25

Question 28

What is the probability that two individuals share 36 bands by chance?

Answer 28

0.25^36= 10^-22

Question 29

What are the assumptions we must make for the probability calculations in DNA fingerprinting?

Answer 29

Two individuals are unrelated Population is homogeneous so results could be biased The detection of 1 allele does not affect the detection of any other allele

Question 30

What is the difference in DNA profiling by Southern Blotting compared to DNA Fingerprinting by Southern Blotting?

Answer 30

Uses single locus probes (SLPs) Only at one locus Uses High stringency conditions The specific loci sequences are known

Question 31

Why are single locus probes very specific?

Answer 31

They only bind to alleles at specific loci and not to related ones

Question 32

Why is the pattern of a DNA profile not truly unique for individual's?

Answer 32

Because fewer loci are used compared to in fingerprinting and the probes are well-characterized so a unique profile is not made.

Question 33

What is used in the standard DNA profiling method?

Answer 33

Short Tandem Repeats using polymorphic microsatellites

Question 34

What does it mean when the loci are highly polymorphic?

Answer 34

They can be found in many alleles (between 5-15 alleles)

Question 35

What are the advantages of DNA profiling using STRs?

Answer 35

Only needs less than 0.1ng of DNA and can use partially-degraded DNA which is useful in crime scenes or when there is little DNA available. Also available as an automation kit. Its sensitivity improves with PCR technology

Question 36

What is CODIS?

Answer 36

Combined DNA Index System. A data system used to rapidly profile alleles in a popolation

Question 37

In the STR loci D21S11 which allele is it found on?

Answer 37

Chromosome 21

Question 38

On a read out using ABI Prism System/ any semi-automated DNA sequencer, what do the: single peaks mean? double peaks mean? taller peaks mean?

Answer 38

Homozygous individual for that gene Heterozygous individual for that gene more repeated number of STRs

Question 39

What are the ethical implications of DNA fingerprinting/profiling databases?

Answer 39

How long is it ethically right to keep DNA samples of individuals in a database? How long is too long?

Question 40

What is AMEL and how is it used for gender typing?

Answer 40

The protein amelogenin is used in making tooth enamel. It is found on the sex chromosomes X and Y but as slightly different lengths (X- 106bp and Y- 112 bp) So in females there is 1 band (XX) and in males there are 2 bands (XY). If its a girl there is 1 band and if its a boy there are 2 bands.

Question 41

How are allele frequencies determined?

Answer 41

By multiplying all the frequencies together.

Question 42

What are some applications of DNA profiling?

Answer 42

Forensics Immigration cases Family pedigrees and family ancestry Species typing in rare breeds

Question 43

What is Ribotyping in bacteria?

Answer 43

Using 16S RNA, we can find specific gene sequences of bacterial species to produce a profile using PCR and Southern Blotting. More specific than identifying bacterial strains from a conventional culture.

Question 44

What is a dendogram?

Answer 44

A diagram that makes it possible to deduce evolutionary divergence of strains and see how they are related.

Question 45

What is mitotyping?

Answer 45

Using mitochondrial DNA, we can produce a profile if there is no genomic DNA available because mtDNA is more robust and survives longer than genomic DNA.

Question 46

What is mitotyping used for?

Answer 46

Used to track human immigration throughout generations.