Topic 5 - Tetrad analysis

Recombination frequency (RF)

used to create linakge map, proportional to distance between 2 genes, indicates relative order and distance

grnr mapping and RF

estimate distance between 2 genes, formula of how often crossing over occurs

RF calculation in diploids

number recombinant progeny/ total progeny

RF in Haploid

only recombinant genotypes, uses Fungal asci (4 haploid product - tetrad or 8 - Octad)

Neurospora (fungi) reproduction

2 mating types, haploid myceliem fuse and form 2n nuclei (diploid stage), undergoes meiosis (4 haploid nuclei), mitotic division forms 8 haploid ascospores (in ascus), linear ordered tetrad

Tetrad analysis

calculate gene-gene distance(linear and unordered), calculate gene centromere distance (need be linear), spore pattern dependent which chromatid undergo crossing over

why recombination can’t be used to map centromere in most eukaryotes

little to no recombination (little heterozygosity), heterochromatin

Centromere mapping conditions (2)

monocentric centromeres (localized kinetochore), retain tetrad together and ordered in ascus (not spread)

Centromere mapping

pay attention to 1st and 2nd divisions

centromere mapping when no crossing over

4:4 pattern, all parental

centromere mapping when crossing over/recombination

2:2:2:2, ½ parentla and ½ recombinant, ½ symmetric and ½ asymmetric after mitosis

2 ways to calculate distance from centromere

RF = (#second division segregation asci/total asci) x ½ (bc only ½ spores recombinant), RF = [(symmetrical second division asci x 2)for asymmetric))/ total] x ½ (bc only ½ recombinant)

3 outcomes of crosses in haploids

Parental Ditype (PD) - all parental, Non Parental Ditype (NPD) - all recombinat, Tetra types (T) - ½ parental + ½ recombinant

Calculation for RF in haploid

RF = (1/2T+NPD)/total, corrected for missed DCO RF = (1/2T+3NPD)/total

Tetrad with linked genes

PD combination preferred - lack of crossing over (or 2 strand DCO), Tetra types less frequent than PD (SCO and 3 strand DCO), NPD least frequent (only from 4 strand DCO)

Gene distance underestimation

large distance underestimated due to invisibles DCO, why 3NPD in correct gene-gene distance formula

Homologous recombination

exchange of genetic material between homologs, takes place in prophase I, accepted model is dsDNA break model

Double stranded break model

Homologous recombination initiated by dsDNA break, not spontaneous but generated breaks, discovered in yeast in 1983

Steps of Homologous recombination

dsDNA break in same location, 5’ end degraded and 3’ overhang created, 3’ end invades other duplex and forms Displacement (D) loop, Invading uses adjacent as template, DNA syntehsis until 5’ end reached, ligation results in Holliday junction, opposite sense resolution

Heteroduplex

where there is DNA from non sister chromatid

Dicovery of Double strand break model

proposed by Szostak/Orr-weaver/Rothstein/Stahl, in yeast

Proteins of homolgous recombination in Yeast

Spo11 initiated with Mrx and Exo1, Rad51 and Dmc1 help facilitate invasion/D loop formations, Rad52 and Rad59 assist formation 1st heteroduplex/Holliday junction, Rad51c-XRCC3 (RuvAB and RuvC in bacteria) resolve holliday junctions

Opposite sense resolution

cutting and rejoinning DNA strands in one holliday junction and outside the other, result in heteroduplex DNA and recombination of flanking gene, more common

Same sense resolution

cutting and rejoinning DNA strand in both holliday junction, no recombination of flanking genes, rare

Gene conversion

process, DNA sequence of one allel altered to another, not completely complementary heteroduplex cause nt mismatch, sometime repaired by mismatch repari (random which is template)

Aberrant ratios

observed ratio of spores that are not expected (6:2 or 5:3), from gene convrsion or spindle overlap

Aberrant ratio 6:2

both strand repaired by using same strand as template

Aberrant ratio 5:3

one strand repaired well other not repaired

aberrant ratio 4:4

no repair of strands (3:1:1:3)

Restriction enzymes discovery

in bacterial cells, protects agaisnt viral infection, Bacterial modifies (methylates) their DNA sequences

Restriction enzymes

recognize specific sites (palindromic 4-8bp), cuts both strand of DNA, sticky or blunt ends

Sticky ends

single stranded overhangs produced by restriction enzymes, can base pair with complementary sequences

Blunt ends

no single strand overhangs produced by restriction enzymes

Probability of cut occuring

(1/4)^n, n = site length

Restriction maps

number and size of fragment produced by digesting with restriction enzymes, depends on size of genome and abundance of each nucleotide, more restriction enzyme leds to more accurate map

Methods for detecting/quantifying DNA/RNA/protein

Southern blot - DNA, Northern blot - RNA, PCR - DNA, Reverse transcription - RNA

Polymerase chain reaction (PCR) requirements

dsDNA template, Nucleotide supply, heat stable DNA polymerase (taq), 2 different primers, buffer solution

Steps of polymerase chain reaction (PCR)

denatures dsDNA at 95C, anneal primers at 55C, extend at 72C, repeat

PCR amplification amount

2^n, n = number of cycles

Primers

short oligonucleotide, complementary to flanking region of target

reverse transcription PCR (RT-PCR) / quantifying PCR

for RNA, convert RNA to DNA (complementary DNA), regular PCR steps, probe is added give fluorescene when new cDNA formed

RT-PCR and fluorescene

level of fluorescene depends quantity of RNA, Ct value

Ct value

number of cycles require for fluorescene signal to exceed threshold, lower value = more RNA

Northern blotting

Detects specific RNA, RNA extracted and purified, separated on gel according to size, blotted onto nylon membrane, probed with labelled DNA, RNA that complementary to DNA detected as labeled bands

Southern blotting

detects specific DNA, DNA extracted and purified, separated on gel according to size, Gel soaked in NaOH to denature, blotted onto nylon membrane, probed are radioactively labelled

Labelled probe

labelled nucleotides, incorporate into region of interest (DNA), membrane washed so only complementary region anneal

Mapping with molecular markers

allow direct detection of difference between individuals, takes into account variation in DNA polymorphisms, typically in noncoding region, inherited in Mendialian fashion (not subject to environment)

Identification of DNA sequence variation

compare sequenc of different organsims, align side by side and note differences

Single Nucleotide Polymorphisms (SNPs)

most common type of sequence differences, one base pair substitued by another, typically in noncoding regions (no detectable genotype effect), if inexpressed region affect phenotypes

Application of SNPs

gene mapping, crime scene DNA analysis, paternity testing

Identification of SNP

genome sequencing, short restriction sequences

Restriction fragment length polymorphisms (RFLP)

Inherited variability in number or length of restriction fragments, in DNA with multiple restriction sequence, when SNP affect restriction site