Topic 4b: DNA Profiling

Question 1

What are the two types of genetic profiling?

Answer 1

Multilocus DNA fingerprinting (minisatellites), and single locus DNA fingerprinting (microsatellites)

Question 2

What does VNTR stand for?

Answer 2

These are variable number of tandem repeats, they are regions of DNA which contain a short nucleotide sequence (core sequence) which is repeated tandemly (in a series) many times.
They are classified as minisatellites (10-60 bp) or microsatellites (2-5 bp) depending on the length of the core sequence
Variation in fragments due to the number of repeat units can be estimated by using the length of PCR products (longer the product, the more repeats, and vice versa)

Question 3

What does multi-locus DNA fingerprinting (minisat) produce?

Answer 3

It produces a pattern resembling an individual specific barcode, it allows for simultaneous detection of alleles at many loci that share a homologous core sequence
You detect the minisatellite using a probe that detects the core repeat DNA sequence, and the alleles differ in length according to their number of repeat units.

Question 4

Can a multi-locus pattern be inferred from a combination of single locus probes? What about vice versa?

Answer 4

YES, you can infer a minisatellite (multi-locus pattern) from many single locus probes (microsatellite), BUT you cannot go the other way, because you cannot determine which bands are alleles.

Question 5

What does a single locus probe show?

Answer 5

It shows homozygotes (one thick band) or heterozygotes (two bands). You can use these to infer multi-locus DNA fingerprinting, but not the other way around

Question 6

How do you interpret DNA fingerprints? What is bandsharing?

Answer 6

You interpret DNA fingerprints via bandsharing. Bandsharing is where relatives share more bands than non-relatives due to identity by descent, non-relatives will share some common bands, but 50% of the bands seen in the offspring must be present in one or the other parent

Question 7

Why does a DNA barcode have different intensity bands?

Answer 7

This depends on hybridization conditions,

Question 8

What are the limitations to minisatellite fingerprinting?

Answer 8

Lots of high quality DNA required
Bands of the same size may be unrelated
Some bands might migrate off the gel
Unknown allelic states
Unknown linkage relationships
Complexity and need to analyze samples in the same gel
Difficult to analyse population genetic markers

Question 9

What is single-locus DNA fingerprinting (microsatellites)?

Answer 9

Core sequence consists of 2-5 nucleotides (therefore much shorter than minisatellites), used extensively in mapping the human genome, now being used for DNA fingerprinting in forensics

Question 10

How do you use restriction enzymes and PCR products to amplify a microsatellite region of the genome?

Answer 10

You design primers that bind to the unique flanking sequence of the region you want and then these will bind up and downstream of the site, thus amplifying only the region of interest. We assume that the flanking sequence does not change

Question 11

What are some reasons why we use microsatellites? (why are they good)

Answer 11

It uses PCR therefore we do not need large amounts of DNA, and this DNA does not need to be high quality (unlike minisatellites)
You can easily find a suite of single locus probes, microsatellites are abundant in the genome
Alleles are described by the number of nucleotides (number of repeats) in the PCR product, we can compare lane to lane or gel to gel, they are codominant

Question 12

What is PCR?

Answer 12

Polymerase chain reaction, relies on thermal cycling there a specific region of DNA is amplified through cycles of denaturation, annealing of primers, and DNA synthesis (each at a different temperature)

Question 13

Why is PCR important for studying populations?

Answer 13

DNA can be amplified from a single gene copy, it is non-destructive for small animals/insects, and specific sequences can be targeted without cloning (mitochondrial genes, nuclear genes and repetitive DNA)

Question 14

How do you develop microsatellites via cloning?

Answer 14

Isolate DNA, digest DNA, clone into vector
Label the repeat sequence probe and isolate the positive clones and then sequence them
Construct pairs of primers complementary to the unique DNA surrounding the repeats
Test primers on a sample from a population of the species

Question 15

What other method can you use to develop microsatellites?

Answer 15

Next generation sequencing where you sequence the genome and look for the repeats, and then design the primers up and downstream of it

Question 16

What is the method used for amplification and detection of microsatellites?

Answer 16

Short length can be effectively amplified by PCR and length determined to the nearest bp (up to 500bp)
Detection is done either radiolabeling (one of the primers) and visualizing it in a polyacrylamide gel, OR fluorescent labeling and an automated analyser.

Question 17

What is a major mechanism of microsatellite mutations?

Answer 17

Slipped strand mispairing during DNA replication, where the polymerase makes a mistake in replication and there is a repeat that buds into the sequence or buds out of it.

Question 18

What are the three different types of microsatellite mutations and their structure?

Answer 18

Di/Tri/Tetranucleotides get added in
Perfect array, imperfect array, compound repeat array (important feature is the length of uninterrupted arrays)
Sequence composition (AT vs GC rich)

Question 19

What are the shortcomings of microsatellites?

Answer 19

Mutation process not fully understood
Some genomes have fewer useful ones
Not always randomly or evenly distributed in the genome
Homoplasy (reversion of a mutation back to ancestral state, hard to detect/ make phylogenetic inferences)
Null alleles do NOT amplify (due to mutation in flanking sequence, so one or both alleles are not amplified, makes heterozygotes look like homozygotes)
Difficulty determining genotype errors due to interpreting stutter vs true bands

Question 20

What are null alleles?

Answer 20

These are non-amplifying alleles, they fail to amplify during PCR therefore they often make heterozygotes look like homozygotes.

Question 21

What are 3 reasons for the occurence of null alleles?

Answer 21

1. Short allele dominance: short alleles get preferentially amplifies, and the long ones drop out
2. Poor quality or low concentration template DNA may result in only one copy priming and amplifying in the early stages of PCR and taking over the reaction
3. Point mutations in the flanking sequence of an allele prevent priming and amplification of some alleles

Question 22

What is one piece of evidence that there may be a null allele at a locus? What evidence is normally used?

Answer 22

Comes from HWE disequilibrium, if there is an excess of homozygotes at a locus.
Usually pedigrees are used, if the known father shares no alleles with an offspring at a locus, there is probably a null allele, thus when primers are designed for different regions of the flanking sequence the father becomes heterozygous and shares an allele with the offspring

Question 23

What are stutter bands?

Answer 23

These bands are caused by slippage and stuttering during PCR. Slipped-strand mispairing during amplification (of polymerase). This makes interpretation of profiles difficult.
This slippage causing some fragments that are made to be shorter than the target sequence by 1-2 units

Question 24

What are the 6 major reasons why microsatellites are used over other methods of population studies?

Answer 24

PCR based, co-dominant, abundance, high mutation/polymorphisms (therefore many alleles per locus), allow cross-species amplification, selectively neutral