Physical and transcript mapping

• Physical mapping

• Transcript identification

Physical and transcript mapping

• Somatic Cell Hybrids–Monochromosomal–Polychromosomal–Radiation hybrids

Physical and transcript mapping

• Somatic Cell Hybrids–Subchromosomal assignment by PCR of

hybrids with different chromosomal content.

Physical and transcript mapping

• Radiation hybrid maps–Random breakpoints. –Ordering of markers.

Physical and transcript mapping

• Fluorescence in situ hybridization–YAC hybridization. –Translocation breakpoint 3q26 and 17q23.

Physical and transcript mapping

• Fluorescence in situ hybridization–Chromosome painting. –Chromosome 8 paint.

Physical and transcript mapping

• Flow sorting chromosomes–Fluorescent dye labeled chromosomes. –Flow karyogram.–Fractionate whole chromosomes Yeast/YACs

Physical and transcript mapping

• Pulsed field electrophoresis–Rotating magnetic field. –Large DNA fragments.

Physical and transcript mapping

• Clone contig–Overlapping DNA fragments.

Physical and transcript mapping

• Chromosome walking–Clone to clone hybridization.

Physical and transcript mapping

• Inverse PCR–Clone flanking unique DNA sequences.

Physical and transcript mapping

• Bubble PCR–Amplification of uncharacterized sequences

flanking known DNA.

Physical and transcript mapping

• PCR based screening–Amplification of row, column and plate pools

for clone identification.

Physical and transcript mapping

• Contig assembly–YAC contig assembly by STS content

mapping.

Physical and transcript mapping

• Inter-SINE PCR–Amplify unique sequences between repeats.–Species specific (Alu in primates).

Physical and transcript mapping

• Zoo blot–Study highly conserved sequences.–Species specific.

Physical and transcript mapping

• Exon trapping

Physical and transcript mapping

• cDNA selection using magnetic bead capture

Genetic mapping

Genetic markers

Recombinants

Genetic vs. Physical distances

Genetic mapping

Marker Number Features

RFLPs >105 diallelic max het 0.5

VNTRs mini >104 many alleles high het

VNTRs mic >105 many alleles high het

SNPs >106 less informative than VNTRs

Genetic mapping

Defining recombinants

Genetic mapping

Single vs. Double recombinants

Genetic mapping

Relationship between genetic and physical maps for human chromosome 19

Genetic mapping

Recognizing recombinants

AD disorder

A) III-6 is recombinant (disorder + A2 allele)

B) III-1 –III-5 are non recomb and III-6 recomb or III-1 – II-5 are recomb and III-6 is non-recomb

C) III-7 and III-8 have disorder but the A1 allele may not be identical by descent

Genetic mapping

LOD score ratio of likelihood linked recombination fraction = theta)/not linked (recombination fraction = 0.5)

Positive in favor of linkage (+3)

Negative scores against linkage (-2)

Curve 1 linkage (Z>3) no recombinants

Curve 2 linkage (Z>3) recombination fraction 0.23

Curve 3 no linkage (Z<-2) for recombination fractions below 0.12

Curve 4 inconclusive

Genetic mapping

Multipoint mappingHighest peaks are most likely locations

Odds in favor of a position are measured by the degree to which the highest peak overtops its rivals

LOD scores dip near markers that show recombinants with the disease

Genetic mapping

Double recombinants suggest errors

Probability of a true double recombinant with markers 5cM apart is small (below 0.05 x 0.05 = 0.0025)

Genetic mapping

Autozygosity mappingMapping for markers that are identical by descent

Congenital deafnessAffected members all homozygous for AFMa052yb5 and D2S158

Gene between AFMb346ye5 and D2S174