Human Mol Gen

Why is using nuclear DNA not the best idea for this practical?

• Recombination during meiosis means not all DNA is inherited equally from all ancestors

What is so special about mitochondrial DNA which makes it good to use for this practical?

• Circular DNA that doesn’t recombine, only inherited from the maternal line so can trace back through generations (almost exact copies from your ancestor)

What part of Mitochondrial DNA are we amplifying and why is it best to study?

The D loop -

• 1000 bp section that is highly variable

• lots of mutations because non coding area of DNA

What is a haplogroup?

related groups of sequences defined by shared mutations which show regional specificty

What DNA is inherited only from the paternal line? How much recombination does it undergo?

Y chromosome

around 5%

What determines how far back you can trace ancestral relationships?

recombination

Do autosomes recombine? What does this mean for their use in ancestry?

Recombine to form unique combinations of microsatellites and SNPs

• Good for when you want to track close family relations over a few generations or identifying individuals

• Bad for tracking relatives over a large number of generations because DNA isn’t shared equally between ancestors

What can we use instead for tracking relationships over many generations through maternal or paternal lines?

Mito DNA and Y chromosome as it does not recombine and change only happens through mutation

Why is it easier to amplify mito DNA?

Theres so much of it

Why is the Y chromosome more difficult to sequence than mito DNA?

• bigger

• Has a lot of repetitive DNA (heterochromatic)

• only 1 copy in each cell

What do we need to figure out if we have a SNP in our DNA?

• A reference sequence

• The sequence you are interested in

What is the reference sequence for mitochondrial DNA to which all other sequences are compared?

Revised Cambridge Reference Sequence (rCRS)

How do you build a haplogroup phylogeny?

Compare mtDNA sequences with rCRS and group together the sequences that share the same mutations. (Start with one they all have and then break it down into smaller groups H, H3, H3f, H3f1)

Why does mitochondrial/ y chromosome haplogroup phylogeny support the Out of Africa theory?

Most of the ancestral/ basal haplogroups are found in africa

Why are mitochondrial haplogroups more diverse than y chromosome haplogroups?

patrilocality- men stay in the same region while the women move around

How do we know homo sapiens, neanderthal and denisovans interbred?

Nuclear genomic sequences (no relatedness between mt DNA or Y chromosomes)

What are 5 features of a good marker for forensic testing?

• High heritability - so that children will most likely inherit mutation

• Marker will identify an individual - Crime or paternity

• Wont change over lifespan

• Analysis can be done with not a lot of starting material

• analysis is easy and cheap

What did DNA fingerprinting by Alec Jeffreys use as a marker?

MINIsatellites

What are the differences between mini and microsatellites

Minisatellites are bigger and less frequent

What are the steps in analysing a minisatellite fingerprint

1. Extract Nuclear DNA

2. Digest

3. Blot

4. Hybridize with Probe

To analyse relationship

5. DNA fragments of offspring should be mixture from both parents

What are the disadvantages of minisatellite markers?

• you need a lot of starting DNA

• Not ideal for forensics because small and degraded samples are common

• Low throughput

Why did people switch to using microsatellites?

• PCR can be used

• Can be multiplexed - multiple analysed at once

How do we multiplex SSRs?

Multiplex PCR will use different primer pairs in the same reaction with different fluorescent dyes to amplify several different PCR products

What are the steps in analysing a microsatellite fingerprint?

1. Extract DNA (ffrom crime scene and suspects)

2. Purify DNA

3. Carry out multiplex PCR

4. Run PCR products on a genetic analyser (capillary electrophoresis)

5. Assign genotypes

Whats the difference between gel and capillary electrophoresis?

• Capillary uses a very long very thin gel

• Because its longer a higher voltage can be used and separation is much quicker

• It has automated detection (using colour of SSRs) and a very high resolution (down to 1 bp)

What are the advantages of using microsatellite fingerprinting?

• Sample DNA doesnt have to be a lot and can be relatively degraded (better for forensics)

• High power of discrimination - can differentiate between individuals

Why couldnt they use y chromosomal DNA to figure out if the skeleton in the car park was King Richard III?

the descendants using the male line were not related to richard.

“While easier to trace genealogically, the male-line is far more susceptible to false paternity than the female line is to false materinity events“