Linkage analysis, gene mapping etc

Question 1

Difference between recombination/crossover pattern in spermatogenesis and oogenesis?

Answer 1

More chiasmata towards telomeres in spermatogenesis

Generally more chiasmata in oogenesis, and more towards centromeres too.

Question 2

What makes a good genetic marker?

Answer 2

Frequent, polymorphic, mendelian inheritance.

SNPs or VNTRs?

Question 3

Phase known pedigree?

Answer 3

3 generations known genotypes for markers.

(so you know which marker allele the disease is originally inherited with and so which children, third generation, are recombinants)

Linkage Phase is the particular combination of alleles of the parent (second generation)

Question 4

Process of identifying disease genes?

Answer 4

Candidate gene approach is an educated guess to identify possible disease causing gene, from existing knowledge connecting known function of gene to phenotype of disease.

Gene-mapping can also narrow down the possible area by testing linkage to marker alleles in known chromosomal locations. (then sequencing or other typing)

Question 5

How to score a RFLP? (restriction fragment length polymorphism, a type of SNP)

Answer 5

Digest the sample with restriction endonuclease, if the restriction target site is present (one allele) then there will be one more variant of restriction fragment length present than if not present (other allele). Identified by southern blotting (adding probe).

Or add primers that flank the restriction site then run PCR, if the site is present then an extra band is seen on gel electrophoresis.

Question 6

How to identify length polymorphism in microsatellites? (simple sequence length polymorphisms) for use as markers.

Answer 6

PCR with primers flanking the microsatellite sequence. Amplifying microsatellite sequence. Then different lengthed variants make different bands on gel electrophoresis. (2 bands for one sample indicates heterozygosity)

Question 7

How to type/identify presence of SNPs?

Answer 7

Oligonucleotide probe hybridisation: Probe with fluorescent end and quencher end (so fluorescence only shows when complementarily bound and so extended)

When SNP is present, probe does not bind as securely (so melting temperature is lower, so incubated above this temperature but below melting temperature of fully complementary DNA)

Question 8

What is a haplotype?

Answer 8

A collection of alleles of closely linked genes on a chromosome that tend to be inherited together statistically.

(this can be useful if the haplotype contains a disease gene and markers)

Or the group of alleles on a chromosome that actually are inherited.

Question 9

Autozygosity mapping?

(must be autozygous so both copies of disease allele will be segregating with the same small haplotype)

Answer 9

A technique for mapping the approximate location of an autosomal recessive disease mutation relative to marker genes/alleles.

Haplotypes are groups of linked alleles typically inherited together, and the overlapping portions of haplotypes (markers which both haplotypes contain) found in diseased (autozygous) individuals indicate where the disease allele is.

Question 10

What does a 50% recombination frequency between 2 loci tell you?

Answer 10

50cM minimum map distance between them, but this is the same as being unlinked, e.g. on different chromosomes.

Question 11

How to determine if 2 loci (a and b) are linked in experimental organisms? (i.e. not humans)

Answer 11

Cross a heterozygote AaBb with a homozygote aabb. (ideally lots of times to create a large progeny population)

If they obey mendels law of independent assortment then the F1 generation will have an equal likelyhood of the 4 genotypes AaBb, Aabb, aaBb, aabb.

But if “linkage disequilibrium” occurs (so there is a unsually high frequency of AB, ab pairing for example then you know that loci A and B are linked)

Question 12

Why is the general rule that 1cM map distance = 1MB physical distance not very reliable?

Answer 12

More crossing over in oogenesis that spermatogenesis.

Less crossing over near to centromeres.

Some specific crossover hotspots exist.

Question 13

How to assess the significance of linkage analysis? (for example in a small family, or multiple small families)

Answer 13

Statistical analysis score called “log of the odds-ratio” LOD score.

Odds ratio is likelyhood of this recombination frequency θ if the genes are linked/vs likelyhood of observed frequency if unlinked!

High LOD score is >3 evidence linked (1000:1 ratio)

Negative LOD score <-2 evidence for unlinked

Inbetween suggests not enough data for statistical significance.

But LOD scores are additive! (so you can add LOD scores between family pedigrees)

LOD scores can even be used for phase unknown pedigrees (where score is calculated for both possible recombination frequencies) Computers do this tricky shit.

Question 14

Role of non-parametric linkage analysis?

Answer 14

Used for gene mapping in Complex, polygenic diseases, with incomplete penetrance.

Uses affected sibling pairs to avoid problem with incomplete penetrance.

Assumption that relatives have disease predisposing alleles in common.

Question 15

What does it mean if a DNA segment is Identical by descent? IBD

Answer 15

It means it arose from the same original mutation.

So siblings shareon average 50% of their sequence identity by descent.

All IBD sequences are Identical by state. (in that they have the same function or identical sequence)

Question 16

What is the expected allele sharing ratio between siblings?

Answer 16

25% chance of sharing 0 alleles

50% chance of sharing 1 allele.

25% chance of 2 alleles.

Diverging from the normal sharing ratio of 1:2:1 suggests linkage of the alleles to the disease gene, as both the siblings are affected and so have a different sharing pattern (higher likelihood of sharing one or more allele if linked). This can be assessed by complicated computerised LOD scores.

Question 17

Difference between Association studies and Linkage analysis?

Answer 17

Linkage analysis looks for genetic similarities in affected family members.

(requires a map of microsatellite markers)

Association studies looks for genetic differences between affected cases and unaffected controls on a population level.

(require a dense map of SNPs)

Question 18

What are tag-SNPs?

(as possible due to limited Haplotype diversity as identified by HapMap project)

Answer 18

SNPs can be grouped into haplotype blocks (separated by recombination hotspots)

If a tag-SNP locus has a particular allele, you can be pretty sure that the next SNP along has a particular allele because the SNPs are linked, in the haplotype block.

This is useful because you then only need to type the tag-SNPs because you can infer the other SNPs’ alleles from them.

Question 19

Why is the HapMap not a perfect resource?

Answer 19

Because it only has 6million of the estimated 10 million SNPs in the human genome.

And rare ‘SNPs’ with a population frequency of less than 1% (so technically not classed as SNPs) are not included, and neither are other forms of genetic or genomic variation like indels or CNV.