Linkage & Genetic Variation Analyses

how do alleles from linked gene assort

if 2 genes are completely linked, the alleles from the parent will assort together 100% of the time

what are parental & recombinant genotypes

parental - the chromosomes of the parent

recombinant - the recombined chromosome of the child

why can we not be sure of the dihybrid & test cross ratios for recombination of alleles that are “linked'“

when genes are “linked” close together on chromosome, it is less likely they will be reassorted independently. This is because it is less likely that chromosome will split during meiosis to separate these genes hence the genes are sorted dependently, and so ratios cannot be determined

what is coupling & repulsion phase for parental alleles

refers to whether the dominant & recessive alleles are on the same or different parental genes

what is recombination frequency

refers to the frequency in which recombinant genotypes appear

• determined by distance between genes

how is recombination frequency (RF) calculated

RF = (number of recombinants/total progeny) x 100

• max RF = 50%

why is max RF 50%

since each meiotic event only involves one of the sister chromatids

• hence even if any participating chromatid underwent recombinant their sister wouldn’t hence 50%

how can recombination frequency help define linkage

• genes with RF < 50% are linked

• genes with RF = 50% are not linked

what is the genetic mapping principle

the RF is correlated with the distance between genes

• map distance = map units (mu) or centiMorgans (cM)

• 1mu/cM = distance that will produce 1% RF

what are double crossovers

refers to 2 crossover events between loci which restores them to parental arrangement

• further 2 genes apart = ↑ chance of double crossover

how can kai-squared test be used for measuring “unlinked” genes

hypothesis are based on gene being linked

• null hyo = variation due to chance, gene are unlinked

• alt. hypo = variation is due to another reason, gene are linked

what are some of the benefits of a trihybrid cross

• faster

• more accurate (tests for double recombinase)

how do we calculate RF from a trihybrid cross

1. consider only 2 genes at a time

2. find all combinations of those 2 genes that match the parental combinations

3. add those together, anything else is recombinant, thus can calculate RF

4. repeat for the other 2 combinations of step 1

what are 2 ways to identify double recombinant progeny for a tricross map

1. the smallest number since double recombination is rare

2. look for combination that are parental on the outside but recombinant in the middle (i.e. a different allele)

how do we take into account double crossovers

(original recombinants + 2 x double recombinants) / total

how can we calculate if interference changes the proportion of double crossovers

chance of DCO =

(chance of crossover between A & B) x (chance of crossover between B & C)

what is the coefficient of coincidence & interference (C)

C = observed/expected

I = 1-C

what are some uses for genetic maps

• Used to identify disease causing rare disease causing alleles

  • Molecular markers mostly used in humans

• Linkage must be considered in genetic counselling/ risk calculations

• Assist in genome sequence assembly

what are some “small variant” DNA changes

SNV (single nucleotide variant) - single change in the nucleotide

indel - insertions / deletions

what are some “large variant” DNA changes

refers to large changes in DNA makeup

• deletion

• duplication

• inversion

• insertion

• translocation

what are different variations of DNA repeats

tandem

• micro-satellites

• mini-satellites

interspersed

• transposons

• retroelements

what is the human reference genome

a standardized sequence comprised of non-pathogenic sequences, used to compare human variations

what is first generation (sanger sequencing)

primer binds to template and ddntps are sued to reconstruct the sequence and thus identify the sequence present

• suitable for confirming small variants

what is second generation sequencing (massively parallel sequencing)

split your sample into millions of small DNA pieces and sequence them all simultaneously in parallel “lanes” or “clusters” on a solid surface or inside tiny wells

what is third generation (long read sequencing)

sequences long strands of DNA by keeping them intact, thus whole genomes can be sequenced in single runs

• good for identifying large structural variants

what are some dis/advantages to sequencing (NGS)

ADV:

• can provide complete information (known & novel variants)

• non-targeted & target (don’t need to know what your looking for)

DIS:

• slower that genotyping

• relatively expensive

what are some dis/advantages to genotyping

ADV:

• faster than NGS

• usually cheap

DIS:

• targeted (need to know what your searching for)

• provides partial information (only known variants)

what is restriction fragment length polymorphisms (RFLPs)

refers to how small variants may change the recognition site for a restriction enzyme

what are 2 methods to identify RFLPs

1. identifying RFLPs using PCR amplification

   • amplify region with restriction site

   • 1 with & 1 without will be produced

2. identifying RFLPs using labeled probes

   • probe binds to restriction site if present

what is a problem with RFLP genotyping

many small variants don’t change restriction cut sites, hence pathogenic changes may not be detected

how can probe hybridization identify alleles

a complementary probe binds to a sequence of nucleotides, if the probe is completely complementary it will have the maximum number of H-bonds, if not completely complementary limited number

hence when we heat them the non-complementary one will dissociate first, thus indicating a variant

how can PCR be used to detect variants via annealing

change the identity of the primers such that they bind if the variant is present

• product changes based on the annealing of the primers

• good for SNVs & indels

what are micro/mini satellite repeats

microsatellite - repeats of 2-10bps

minisatellite - repeats of 10-100bps

how can micro- & minisatellite repeats be detected

detected based on the length of the product

• gel electrophoresis separates based on length