1. Genomes Third Edition Chapter 3: Mapping Genomes Copyright © Garland Science 2007 Terry Brown

2. Chapter Objectives  Why mapping is important before sequencing of the genome?  What are genetic and physical maps?  Learning the techniques to construct genetic map of a genome  The role of linkage analysis in map construction  Learning the techniques to construct physical map of a genome

3. Objectives of Today’s Lecture  Importance of genome mapping before sequencing of the genome?  Understanding the way genome are mapped  Genetic Mapping Linkage Analysis

4. Importance of genome mapping before sequencing of the genome?

5. Mapping Genomes  Genome sequencing methodology depends on sequencing technology available.  Even the most sophisticated techniques available now can sequence about 750bp in a single experiment.  So we need to construct the sequence of long DNA molecules from a series of shorter sequences.  By breaking the molecule into fragments and then determining the sequence of each one.  Then assemble the sequence by searching over lap and then build up the master sequence.  This method is known as shotgun method.

6. Mapping Genomes  The Shotgun method works very well with the small prokaryotic genomes.  The complexity of analysis increases disproportionately when large genomes are analyzed. The number of possible over laps increases when number of fragments increases like: 2n 2 -2n for 2 fragments we there would be 4 possible over laps while for 4 fragments where would be 24! The problems with reparative sequence

7. Mapping Genomes  So the shotgun sequencing technique can not be applied solely in sequencing of larger eukaryotic genomes.  Rather a genome map is generated first which provide a road map for sequencing project and help in assembly of genome sequence.  The genetic map provides gene position and other distinctive features.  After having genome map the sequencing can proceed in either of the two ways: Whole-genome shotgun method Clone contig method

8. Figure 3.3 Genomes 3 (© Garland Science 2007)

9. Understanding the way genome are mapped

10. Genetic and Physical Maps  The Genetic Map:  Based on the use of genetic technique to construct maps showing the position of genes and other sequences features on a genome.  Obtained by linkage analysis using cross-breeding experiments and family histories (pedigrees)  Physical Maps:  Uses molecular biology techniques to examine DNA molecules directly in order to construct map showing the positions of sequence features, including gene.

11. Genetic and Physical Maps  The Genetic Maping:  Based on Markers (Land marks) Important genes Biochemical markers DNA markers  Restriction Fragment Length Polymorphisms (RFLPs)  Simple Sequence Length Polymorphisms (SSLPs)  Single Nucleotide Polymorphisms (SNPs)  Linkage analysis of Markers (enable positioning of land marks) Based on recombination frequencies of genes/markers

12. Genetic Maps  The genetic map shows the genetic markers as land map shows distinctive features on landscape like rivers, road and building.  Genes were the first genetic markers  The early map constructed during early twentieth century were of genes having two alternative forms i.e. alleles with different phenotypes.  These genes were those with can be recognized by eye i.e. pea pod color, height of plant, shape of wing of fruit fly. Etc  The limitation of observable characters and to map those organisms which have few visible characters like microbes, The need of other markers were soon realized: Biochemical markers ABO blood groups HLA typing system  (HLA-DRB1=290 alleles  HLA-B= >400 alleles)

13. DNA Markers for Genetic Mapping  Using gene as a marker is very useful but it has limitation.  Gene occupy very small portion/space of genome and are not evenly distributed in the genome.  And also every gene not have allelic forms or can not distinguishable easily.  Therefore the map based on gene is not detailed and comprehensive.  So other features which were not a gene, were used as marker and known as DNA markers Restriction fragment length polymorphisms (RFLPs) Simple sequence length polymorphisms (SSLPs) Single nucleotide polymorphisms (SNPs)

14. RFLP  Based on recognized restriction sequence length EcoRI (4 6 = 4096)  Density : 10 5 RFLPs in a mammalian genome. 

15. Simple Sequence Length Polymorphisms (SSLPs)  SSLPS are array of repeat sequences that display length variations i.e. different alleles containing different numbers of repeat units.  They can be multiallelic i.e. each SSLP can have a number of different length variant.  They are Minisatellite or variable number of tandem repeats (VNTRs) Repeat unit is up to 25bp in length (not evenly distributed found at ends)  Microsatellite or simple tandem repeats (STRs) Repeat unit are 13bp or less (10-30 copies 6bp) (5x10 5 micro >6bp in human)

16. Single Nucleotide Polymorphisms (SNPs)  The individual of a species have genome which differ at many nucleotides positions. i.e. A in one person and G in other.  Some of these may give rise to RFLPs  There are about 4Millions SNPs in human genome (one SNP per 10kb of eukaryotic genomes).  Theoretically each SNPs should have four alleles but most of SNPs are biallelic ?????  The SNPs are scoured by oligonucleotide hybridization analysis  DNA chips  Solution hybridization techniques

17. Single Nucleotide Polymorphisms (SNPs)

18. DNA microarrays and Chips  DNA microarrays: Target sequences are spotted onto a glass or nylon membrane of 18x18mm into 80x80=6400 spots  DNA chips: DNA target sequence are synthesized by photolithography onto a wafer of glass or silicon with high density about 300,000 oligos per cm 2

19. Hybridization with an oligonucleotide with a terminal mismatch

20. Oligonucleotide ligation assay (OLA) & Amplification refractory mutation system (ARMS)

21. Linkage analysis is the basis of genetic mapping  Principles of Inheritance: Law of random segregation of alleles Law of independent segregation of pairs of alleles.  Genes on the same chromosomes should inherent together  Linkage  Partial linkage

22. Figure 3.14 Genomes 3 (© Garland Science 2007)

23. Figure 3.15 Genomes 3 (© Garland Science 2007)

24. Figure 3.16 Genomes 3 (© Garland Science 2007)

25. From partial linkage to genetic mapping  Morgan and his student Arthur Sturtevant proposed the frequency of recombination is a measure of distance between two genes.  This can be used to construct the order and map of genes along the chromosome.  Limitations Recombination hot spot Double cross overs

26. Linkage analysis with different types of organisms  Linkage analysis with species such as fruit flies and mice, WHERE PLANNED BREEDING EXPRERIMENTS ARE POSSIBLE  Linkage analysis with humans, WHERE PLANNED BREEDING EXPRIMENTS ARE NOT POSSIBLE  Linkage analysis with bacteria, WHERE MEIOSIS DOES NOT OCCURE.

27. Linkage analysis with planned breeding experiments  First developed by Morgan and his colleague.  Based on recombination frequency calculation with the breeding experiments. i.e. with fruit flies.  Possible for all eukaryotic systems including humans.  Ethical limitations narrows the scope of this technique for humans???  Based on observable markers as well as DNA markers i.e. RFLPs, SSLP and SNPs.  Use of DNA marker also makes possible the direct observation of games i.e. sperms and ovum to calculate recombination frequencies.

28. Linkage analysis by using human pedigree  With humans its difficult to plan breeding experiments therefore for finding the recombination frequencies we need to base on the genotypes of available marriages and their off springs in the pedigree.  The direct observation of sperms is also possible but is difficult.  Example of pedigree analysis of association of gene and a maker M with four alleles.  Association of linkage is established with LOD score (Logarithm of the odds)

29. Genetic analysis in Bacteria  Meiosis is not there in bacteria but they do exchange genetic material which can recombine.  The recombination frequencies can be calculated for these recombination if genetic difference (mutation) are associated with some phenotypes.  Genetic exchange occurs by:  Conjugation Complete chromosome or part of it can be transferred by conjugation tube. Chromosomal DNA can be transferred by episome transfer integrated in plasmid (up to 1MB)  Transduction By bacteriophage (50kb)  Transformation Direct from environment (less than 50kb)

31. Physical Mapping  Genetic map alone is rarely sufficient for directing the sequencing phase of genome project.  Resolution: The resolution of genetic map depends on the number of crossovers that have been scored. This is easy with bacteria and small eukaryotes which can be grown in huge number so many crossovers can be observed enabling the construction of highly detailed genetic maps. E. coli genome sequencing project in 1990s, the genetic map contained 1400 markers (average 1maker per 3.3 kb). Saccharomyces cerevisiae project (1150 makers 1 per 10kb). But with humans large number of progeny can not be obtained so few cross over can be studies. The genetic map is not finely resolved i.e. genes several kilobases apart may appear at same position on the genetic map  Inaccuracy: Crossovers are not randome i.e. recombination hot spot etc

32. Physical Mapping  We need to SUPPLEMENT and RECHECK the Genetic map with other techniques such as Physical mapping.  Physical mapping can be done with: Restriction mapping Fluorescent in situ hybridization (FISH) Sequence tagged sites (STS) mapping

33. Restriction mapping  We can utilize unique cutter for restriction mapping.  We can map the restriction site in a DNA up to 50 kb of size using double restriction and partial restriction.  The resolution limit can be enhanced by using special Gel electrophoresis techniques i.e. Orthogonal field alteration gel electrophoresis (OFAGE).

35. Direct observation of DNA molecules for restriction sites  We can directly observe the restriction site on chromosomal DNA by:  Gel Stretching  Molecular combing

36. Fluorescent in situ hybridization (FISH)  FISH enables the position of a marker on chromosome or extended DNA to be directly visualized.  By hybridization with fluorescent probe.  The FISH can also be applied to genome clone library, enabling the mapping of clones to genome map.  FISH can be done with Mechanically stretch chromosomes Centrifugation based 20X stretching (resolution 200-300 kb)  Nonemetaphase chromosomes Interphase chromosomes (resolution 25kb) Fiber FISH (10kb) (Stretching of interphase chromosome by gel stretching or molecular combing)

37. Sequence tagged site mapping  To generate a detailed physical map of a large genome we need high resolution and high throughput techniques. Restriction mapping can not be applied on large genomes FISH provide detailed mapping but takes much time and require huge experimentations.  Presently the most powerful physical mapping technique of large genomes is STS mapping.  STS is simply a short DNA sequence generally 100 to 500 bp and occurs only once in the chromosome or genome.  STS mapping is performed by multiple STS or set of STS on broken/fragmented chromosome/genome.  A collection of DNA fragments is madee by isolating a chromosome and then breaking it into smaller pieces, so that in collection a single point can be represented about five/six times.

38. Sequence tagged site mapping  The mapping is performed by amplification of STS unique sequence using PCR and looking for the presence of two different STS on the same fragment from the collection.  The frequency of having two STS on the same fragment depends how close they are to each other.  Closer the STS to each other higher the chance to find them together on the more fragments.  Or frequency at which breaks occur between two markers.

39. Any unique DNA sequence can be used as an STS  The STS can be any sequence which: Have known sequence Should have unique position in chromosome/genome  The most common STS are: Expressed sequence tags (ESTs) (taken from cDNA projects: limited to genes only) Simple sequence length polymorphism (SSLPs) (mini and micro satellite )  Help in directly linking the genetic and physical map Random genomic sequences 

40. Fragment of DNA for STS mapping  For STS mapping fragment of a chromosome or genomic DNA are needed, which are known as mapping reagents.  These can be produced in many ways:  Radiation hybrids The genome can fragmented by irradiation by X-ray (3000-8000 rad) and then can be fused to make radiation hybrids with hamster cells A single chromosome can be separated by technique like flow cytometry and can be used to make radiation hybrids  A genomic clone library This can be directly mapped with STS and provide a direct linked with STS mapping and then can be sequenced.