Gene mapping methods

This presentation contains information about gene mapping and gene mapping methods. Both classical and molecular methods are being discussed here.

1. Gene Mapping Methods Presented by- Meenakshi Das MSc., PGDBI

2. Introduction Gene mapping means the mapping of genes to specific locations on chromosomes. It is very important in the understanding of genetic diseases. Such maps indicates the positions of genes in the genome and also distance between them.

3. History In 1911, by Thomas Hunt Morgan, gene for eye- color was located on the X chromosome of fruit fly. (1) E.B. Wilson attributed the sex-linked genes responsible for color -blindness and hemophilia in human beings to be located on the X-chromosome, similar to the many X-linked factors being described by the Morgan group in flies.

4. Types Of Gene Mapping Genetic mapping Based on the use of genetic techniques to construct maps. These maps show the positions of genes and other sequence features on a genome. Also helps to determine the relative position between two genes on a chromosome. Physical mapping Uses molecular biology techniques to examine DNA molecules directly. Based on these techniques map construction is done. These maps show the positions of sequence features , including genes.

5. Genetic Mapping A genetic map must show the positions of distinctive features. Requires informative markers – polymorphic and a population with known relationships Best if measured between “close” markers. Unit of distance in genetic maps = centiMorgans , cM 1 cM = 1% chance of recombination between markers

6. Genes-The first marker to be used First genetic map was constructed in early 20 th century. Genes were the first markers used. The first friut fly map showed the positions of genes. For example- body colour, eye colour etc. All these maps were based on phenotype of the organism. But visual phenotypes were limited and even a single phenotype could be affected by more than one genes. Therefore more comprehensive and less complex characteristics were required.

7. Use of Biochemical Markers For microbes and humans, biochemical phenotypes were preferred. A big advantage is that relevant genes have multiple alleles. For example- Gene called HLA-DRB1 has at least 290 alleles and HLA-B has over 400 alleles.

8. DNA Markers for Genetic Mapping Genes are useful markers but not ideal. Mapped feature that are not genes are called DNA markers. DNA markers must have at least two alleles to be useful. DNA sequence features that satisfy this requirement are- Restriction Fragment Length Polymorphism (RFLP) Simple Sequence Length Polymorphism (SSLP) Single Nucleotide Polymorphism (SNP)

9. RFLP RFLP is the first type of DNA marker to be studied. Restriction enzymes cut DNA at specific recognition sequences. But restriction sites in genomic DNA are polymorphic and exists as two alleles. The RFLP and its position in the genome map can be worked out following the inheritance of its alleles.

10. RFLP

11. Scoring an RFLP There are two methods for scoring an RFLP Southern hybridization PCR

12. Southern Hybridization

13. PCR

14. Simple Sequence Length Polymorphism (SSLP) SSLPs are arrays of repeat sequences that display length variation. Here different alleles contain different number of repeat sequences. SSLPs can be multiallelic . Two types of SSLPs are- Minisatellites (VNTRs) Microsatellites (STRs ) Microsatellites are more popular than minisatellites as DNA markers.

15. Typing of SSLP (STRs) by PCR

16. Single Nucleotide Polymorphism (SNP) There are some positions in the genome where some individuals have one nucleotide while others have another. Some SNPs give rise to RFLPs but many do not. SNPs originate when a point mutation occurs in a genome converting one nucleotide to another. There are just two alleles-the original sequence and the mutated version. SNPs enable very detailed genome maps to be constructed.

17. SNP

18. Typing method of SNPs These are mainly based on oliginucleotide hybridization analysis. These are- DNA Chip Technology Solution Hybridization Oligonucleotide Ligation Assay Amplification Refractory Mutation Assay (ARMS Test)

19. DNA Chip Technology Image Source: Internet

20. Solution Hybridization Technology

21. Oligonucleotide Ligation Assay Image Source: GENOMES 3

22. Amplification Refractory Mutation Assay (ARMs Test) Image Source: GENOMES 3

23. Linkage analysis is the basis of genetic mapping Chromosomes are inherited as intact units, so it was reasoned that the alleles of some pairs of genes will be inherited together because they are on the same chromosome. This is the principle of genetic linkage, Pairs of genes were either inherited independently, as expected for genes in different chromosomes, or, if they showed linkage, then it was only partial linkage: sometimes they were inherited together and sometimes they were not T he frequency with which the genes are unlinked by crossovers will be directly proportional to how far apart they are on their chromosome. The recombination frequencyis therefore a measure of the distance between two genes If you work out the recombination frequencies for different pairs of genes, you can construct a map of their relative positions on the chromosome

24. The LOD Score The LOD score often used for linkage analysis in human populations, and also in animal and plant populations. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers). The method briefly, works as follows: Establish a pedigree Make a number of estimates of recombination frequency Calculate a LOD score for each estimate The estimate with the highest LOD score will be considered the best estimate

25. LOD Score The LOD score is calculated as follows: LOD = Z = Log10 probability of birth sequence with a given linkage probability of birth sequence with no linkage By convention, a LOD score greater than 3.0 is considered evidence for linkage. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage.

26. Genetic Mapping In Bacteria Transformation Conjugation Transduction

27. Demerits of Genetic mapping techniques A map generated by genetic techniques is not much sufficient for directing the sequencing phase of a genome project. This is for two reasons : The resolution of a genetic map depends on the number of crossovers that have been scored . Genetic maps have limited accuracy.

28. Physical Mapping The most important techniques used in physical mapping are as follows: Restriction Mapping Fluorescent in situ Hybridization (FISH) Sequence Tagged Site (STS) Mapping

29. Restriction Mapping Restriction mapping locates the relative positions on a DNA molecule of the recognition sequences for restriction endonucleases. The simplest way to construct a restriction map is to compare the fragment sizes produced when a DNA molecule is digested with two different restriction enzymes that recognize different target sequences.

30. Restriction Mapping Image Source: GENOMES 3

31. Limitations of Restriction Mapping Restriction maps are easy to generate if there are relatively few cut sites for the enzymes being used. Restriction mapping is more applicable to small rather than large molecules. In practice, if a DNA molecule is less than 50 kb in length it is usually possible to construct an unambiguous restriction map for a selection of enzymes with six-nucleotide recognition sequences . This problem can be solved to some extent by choosing enzymes which are expected to have infrequent cut sites in the target DNA molecule. These “Rare cutters” fall in two categories: Enzymes with seven-or eight-nucleotide recognition sequences Enzymes whose recognition sequences contain motifs that are rare in the target DNA

32. Direct examination of DNA molecules for Restriction sites Two ways of doing this: Gel stretching Molecular Combing

33. Gel Stretching Image Source: Internet

34. Molecular Combing Image Source: GENOMES 3

35. Fluorescent in situ hybridization (FISH) FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualized . In optical mapping the marker is a restriction site and it is visualized as a gap in an extended DNA fiber . In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.

36. Fluorescent in situ hybridization (FISH) Image Source: GENOMES 3

37. Sequence tagged sites (STS) STS mapping is the most powerful physical mapping technique. Detailed Maps are generated by STS mapping. A sequence tagged site (STS) is a short DNA sequence, generally between 100bp and 500bp in length. STS is easily recognizable and occurs once in the chromosome or genome being studied.

38. Mapping of STS For mapping a set of STSs, a collection of overlapping DNA fragments from a single chromosome or from entire genome is needed. The data from which maps will be derived are obtained by determining fragments which contain STSs. This can be done by hybridization technique and PCR. PCR is preferred because it is easier.

39. Mapping of STS If the two STSs are very close then there is always a chance that they will be on the same fragment. If they are farther apart then they may sometimes be on the same fragment but sometimes they will not. The data can therefore be used for calculating mapping distance between two markers as in linkage analysis. Image Source: GENOMES 3

40. Ways to obtain STSs STSs can be obtained in many ways but the most common methods are: Expressed Sequence Tags (ESTs) SSLPs Random genomic sequences

41. Genetic Map VS Physical Map A genetic map is constructed using recombination frequency calculated from the progenies whereas physical mapping pertains to locating the position of DNA sequences. A genetic map is an indirect method of locating the positions of genes or DNA markers whereas physical mapping is a direct method. The unit of measurement of map distance in genetic map is cM whereas in physical map is the base pair.

42. Importance of Gene Mapping Gene map is the anatomy of human genome . It helps in analysis of the heterogeneity and segregation of human genetic diseases . It helps to develop methods for gene therapy . It provides clinically useful information about linkage

43. References Brown , T. A. (2007) GENOMES 3. 3 rd Edition, Garland Science Publishing Kant, Anil. (2008). Singh, R.K. Mishra G. P., Kant Anil, Shashi Bala . Molecular markers in Plants. In: Molecular Plant breeding; Principles and Applications. pp 79-96 Stadium press LLC, Texas, USA.. 77-79. Morgan, T. H. Random segregation versus coupling in Mendelian inheritance.  Science   34 , 384 (1911) Zamir , D. & Tanksley , S.D . (1988) Mol Gen Genet 213 : 254.

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