1. MAPPING GENOMES – genetic, physical & cytological maps Genetic distance (in cM) 1 centimorgan = 1 map unit, corresponding to recombination frequency of 1% 1 cM corresponds to ~ 1 Mbp DNA for human genome Gibson Fig. 1.3 Physical distance (in bp) - determined by DNA sequencing (or restriction fragment sizing)

2. - no absolute relationship between genetic & physical distances Fig. 3.25 Discrepancies (at times) between genetic & physical maps Genetic mapping data may have limited resolution or limited accuracy (eg recombinational “hotspots”) (eg if not enough progeny) Yeast chr III or “coldspots”

3. STRATEGIES USED IN GENOMIC ANALYSIS Fig. 3.3 Markers used to anchor assembled sequences on map - because of recent advances in rapid sequencing technologies & powerful bioinformatics tools, “shotgun” approach is now usually used - except for genomes with high amounts of repetitive DNA

4. RFLPs – restriction fragment length polymorphisms SSLPs – simple sequence length polymorphisms SNPs – single nucleotide polymorphisms DNA MARKERS USED IN GENETIC MAPPING DNA marker must have (at least) two different alleles to be useful in monitoring inheritance patterns Alleles – alternative forms of a gene (or DNA sequence) at a particular locus (chromosomal site) Polymorphisms – occurrence of two or more variants (alleles, phenotypes, sequence variants) at significant frequencies in a population if present < 2% in population, called “mutation” or “mutant allele” Haplotype – set of alleles linked on a chromosome usually inherited together as a block, “haploid genotype”

5. 1. RFLPs (Restriction Fragment Length Polymorphisms) Fig. 3.4 - inherited pattern of restriction sites follows Mendelian rules

6. How to detect RFLPs? i. by Southern hybridization analysis Fig. 3.5 - if “polymorphic” restriction site is present, detect two hybridizing fragments, vs. one if site is absent Length of large fragment = sum of two small ones

7. Fig. 3.5 ii. by PCR analysis Lane 1 = size markers Lane 2 = DNA homozygous for allele 1 Lane 3 = DNA homozygous for allele 2

8. Fig. 3.6 2. SSLPs (simple sequence length polymorphisms) Microsatellites - tandem repeats of short stretches ( < 5 bp or so) in clusters usually < 150 bp Different number of GA copies in tandem

9. Fig.3.5 - small fragments migrate more rapidly than large ones towards positive electrode Electrophoretic mobility - detect length differences Interpretation if lane A = DNA from person #1 and lane B = DNA from person #2 ? How to detect SSLPs by PCR

10. Fig. 7.24 Differences in copy number in microsatellite array among individuals useful in genetic profiling Blue, Green: fluorescent tagged PCR products Red: DNA size markers - high heterozygosity of microsatellites (multiple alleles in population) homozygous vs. heterozygous state for a particular microsatellite locus? Each lane = DNA profile (with microsatellite length variants) from different individual - estimated that ~ 3 million differences between any two copies of human genome (natural sequence variation) DNA fingerprinting, forensic analysis

11. Fig. 3.7 3. SNPs (single nucleotide polymorphisms) SNPs usually exist as just two variants in population, because for a third allele to arise, mutation must occur at exactly the same position (see p.69)

12. “...found population-specific allele frequency changes.... one SNP in EPAS1 (transcription factor gene involved in response to hypoxia) showed a 78% frequency difference between Tibetan [high altitude] and Han [sea level] samples.” Yi et al. Science 329:75 (July 2 & August 13, 2010) “Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.” Exome = “coding sequences of 92% of human genes”

13. Fig. 3.8A - “stringency” of hybridization conditions (eg.temperature, salt…) influences stability of imperfect complementarity - reduced stability of hybrid between oligomer probe & test DNA if any mis-matches Oligomer-specific hybridization tests How to detect SNPs

14. (i) Detecting hybridization by “dye-quenching” - when oligomer probe forms stable hybrid with template DNA (vs. hairpin structure), generates fluorescent signal Fig.3.8B “molecular beacon” FRET (fluorescence resonance energy transfer)

15. (ii) Detecting oligomer hybridization using DNA microarrays “Oligo chips” Tech.note 3.1 - for large scale analysis of SNPs (see later Topics for other applications) - grid of different oligomers synthesized in situ on glass wafer by photolithographic process - fluorescently labelled DNA (or cDNA…) allowed to hybridize - hybrids monitored by laser scanning - density ~ 10 6 oligos/cm 2 “~150,000 polymorphisms can be typed in a single experiment...” p.71 Affymetrix's GeneChip

16. Griffiths p.404 Note: microarray Fig.T3.2 (p.71) shows RNA expression data not SNP data Why might some signals be stronger than others in SNP analysis?

17. Advantages of DNA markers - easy to generate large number & easy to detect - codominant, can detect both alleles in heterozygote - SNPs & microsatellites rather evenly spread in genome Fig. 3.19 & 3.21 Fig.7.24 Microsatellite analysis by PCR Genetic markersDNA markers

18. PHYSICAL MAPPING APPROACHES 1. Restriction mapping - determine relative positions of restriction sites in DNA molecule by analyzing sizes of fragments generated by restriction enzymes Fig. 3.27

19. 2. FISH (fluorescence in situ hybridization) - intact metaphase chromosome (as ss DNA, denatured with formamide) hybridized with fluorescently labelled probe Fig. 3.32 Drosophila centromere-specific tandem repetitive DNA probe ~ 1 Mbp resolution of markers

20. 10 – 25 kb resolution Fig. 3.31 Fibre-FISH Probes: cosmid 1, cosmid 2, (overlap region in yellow) Nature Feb. 2001 - mechanically-stretched chromosomes or “molecular combing” of isolated DNA for higher resolution

21. 3. STS (sequence tagged site) physical mapping What are STSs? - any short, known DNA sequence (100-500 bp) which is unique in genome - wish to have many STSs along chromosome STSs A B C D E F G HI J K L MN O P Q R S T U V W BACs - if STS is present in two clones, can deduce that those clones are overlapping … so can be used as physical mapping marker

22. 1.ESTs – expressed sequence tags (from cDNA clones, ie. representing mRNAs for various genes see Fig.3.36) 2. SSLPs (Microsatellites) 3. Random genomic sequences Examples of sequences that can serve as STSs - tandem simple sequence repeats with unique flanking sequences as long as single copy in genome eg. only one hybridization signal in Southern experiment

23. Distance between 2 STSs will determine whether they are: likely, sometimes or never located on same DNA fragment In genomic analysis, want overlapping set of DNA fragments spanning genome (or chromosome separated by flow cytometry, Fig.3.38) Fig. 3.35 (eg. clone library)

24. Fig. 3.39 1. Clone libraries Fig. 3.1

25. Potential problems in mapping genomes with repetitive DNA Fig.3.2A - omission of internal regions of tandem repeat array… (DNA sequences shown as single stranded, eg. from computer file) 1. Tandem repeats 2. Dispersed repeats Fig. 3.2B - omission of sequences located between two repeats or incorrect linking (eg from different chromosomes)