DNA Fingerprinting/Profiling Flashcards
What is DNA fingerprinting/profiling?
A technique of determining individual’s characteristics which will be as unique as a fingerprint.
Give some applications of these techniques?
Microbiology Zoology Forensic Science Paternity testing Immigration disputes
What are the repetitive sequence of DNA (that are non-coding regions) known as?
Tandem Repeats and Dispersed Repeats
What are the 3 categories of tandem repeats?
Satellites
Microsatellites
Minisatellites
What is high copy number DNA?
They are highly repetitive short DNA sequences
AKA Satellite DNA
Composed of unequal ratios of AT/CG
Composed of long tandem repeats in heterochromatin
What repetitive sequences with high copy numbers are found in the centromere of the dividing cell?
Alpha satellite DNA and Beta satellite DNA (both vary in length of bps)
What is the structural function of repetitive DNA in telomeres?
Stop the chromosomes from sticking together and maintains the integrity of the chromosome while dividing.
Why do we have repetitive DNA if they don’t make any contribution to phenotype (they are non-coding)?
Because they have just increased in number and abundance over evolutionary time.
Where are repetitive sequences/satellite DNA found in the genome?
In the telomeres, centromeres and throughout the the chromosomal DNA.
Describe how alpha satellite DNA is arranged?
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.
What are the sub-classes of Satellite DNA?
Mini and Micro satellite DNA
How long is microsatellite DNA and where can it be found in the genome?
Typically less than 100 bp long including short tandem repeats (STR) and simple sequence repeats (SSR)
Found throughout the genome
What is microsatellite DNA used for?
Modern DNA profiling
How long is minisatellite DNA and where can it be found in the genome?
Typically 0.1-20kb LONG
Found near telomeres
What is minisatellite DNA used for?
DNA fingerprinting
Explain the principle/idea behind DNA fingerprinting
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
What are the two polymorphisms of satellite DNA?
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)
What are VNTRs?
Short nucleotide sequences (10-100bp) repeating in clusters
Each variant of this acts as an inherited allele
How do we use VNTRs to distinguish between individuals?
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)
How was DNA fingerprinting first discovered and used?
Dr.Jeffreys was studying myoglobin and accidently observed varying DNA of individuals using restriction fragment length polymorphism.
What does high stringency mean in terms of southern hybridisation analysis?
The conditions you use in order to get more specific binding of the probe to the DNA sequence.
How does Restriction Fragment Length Polymorphism (RFLP) analysis work?
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
What are the steps of Southern Hybridisation/DNA Blotting?
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.
When is the banding pattern different in a DNA fingerprint?
Different in different individuals so different sources of DNA used
How can we increase the likelihood that an individual’s sequence is unique to them?
Look at more than one repeating sequence on different loci in the genome
What are the 2 variants of the 16 bp core of myoglobin probe?
Hint: they are multilocus probes
33.6 and 33.15
What is the probability that two individuals would share a fingerprinting band by chance?
0.25
What is the probability that two individuals share 36 bands by chance?
0.25^36= 10^-22
What are the assumptions we must make for the probability calculations in DNA fingerprinting?
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
What is the difference in DNA profiling by Southern Blotting compared to DNA Fingerprinting by Southern Blotting?
Uses single locus probes (SLPs)
Only at one locus
Uses High stringency conditions
The specific loci sequences are known
Why are single locus probes very specific?
They only bind to alleles at specific loci and not to related ones
Why is the pattern of a DNA profile not truly unique for individual’s?
Because fewer loci are used compared to in fingerprinting and the probes are well-characterized so a unique profile is not made.
What is used in the standard DNA profiling method?
Short Tandem Repeats using polymorphic microsatellites
What does it mean when the loci are highly polymorphic?
They can be found in many alleles (between 5-15 alleles)
What are the advantages of DNA profiling using STRs?
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
What is CODIS?
Combined DNA Index System. A data system used to rapidly profile alleles in a popolation
In the STR loci D21S11 which allele is it found on?
Chromosome 21
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?
Homozygous individual for that gene
Heterozygous individual for that gene
more repeated number of STRs
What are the ethical implications of DNA fingerprinting/profiling databases?
How long is it ethically right to keep DNA samples of individuals in a database? How long is too long?
What is AMEL and how is it used for gender typing?
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.
How are allele frequencies determined?
By multiplying all the frequencies together.
What are some applications of DNA profiling?
Forensics
Immigration cases
Family pedigrees and family ancestry
Species typing in rare breeds
What is Ribotyping in bacteria?
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.
What is a dendogram?
A diagram that makes it possible to deduce evolutionary divergence of strains and see how they are related.
What is mitotyping?
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.
What is mitotyping used for?
Used to track human immigration throughout generations.
