Gene mapping and cloning of disease gene

gene mapping

1. GENE MAPPING AND CLONING OF DISEASE GENE BY DINESH K M PHARM PHARMACOLOGY

2. GENE MAPPING INTRODUCTION Gene- a unit of heredity which is transferred from a parent to offspring and is held to determine some characteristic of the offspring. The first marker to be used. MAPPING- determining the location of elements within a genome with respect to identifiable landmarks.

3. Types of mapping Genetic mapping- linear description of DNA markers/genes on a given chromosome with closely placed markers being inherited together more often. Physical mapping- physical location on the chromosome, relating more towards exact positioning of gene elements. Comparative mapping Genome/genetic maps – graphic representation of the relative positions of genes a DNA sequences.

4. Genetic mapping Linkage mapping Pedigree Polymorphic markers Physical mapping Cytogenetic mapping Somatic cell mapping Radiation hybrid mapping Restriction mapping- PFGE,BAC contigs , sequencing Comparative mapping Gene sequences Data bases DNA chips

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. 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 requirements are- -restriction fragment length polymorphism(RFLP) -simple sequence length polymorphism (SSLP) -single nucleotide polymorphism(SNP)

7. 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.

8. Two methods of RFLP Southern hybridization PCR Polymorphic restriction site DNA (ALLELE 1) DNA (ALLELE 2) add the restriction endonuclease 4 fragments 2 fragments

9. 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 b multiallelic. Two types of SSLPs are- -Minisatellites(VNTRs) -Microsatellites(STRs) Microsatellites are more popular than minisatellites as DNA markers..

10. Single nucleotide polymorphism(SNP) There are some positions in the genome where some individuals have one nucleotide while others have another. Some SNPs give to RFLPs but many do not. SNPs originate when a point mutation occurs in 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.

11. Typing method of SNPs These are mainly based on oligonucleotide hybridization analysis. These are- -DNA chip technology -solution hybridization -Oligonucleotide ligation assay - A mplication refractory mutation assay (ARMS test)

12. LOD score The LOD score used for linkage analysis in human populations, and also in animal and plant populations. Computerized LOD score analysis is a simple way to analyse complex family pedigrees in order to determine the linkage between mendelian traits. The method briefly, works as follows: -establish a pedigree -make a no 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.

13. LOD Score It is calculated as follows: LOD=Z=log 10 probability of birth sequence with a given linkage probability of birth sequence with no linkage greater than 3.0- evidence for linkage less than 3.0 – evidence to exclude linkage

14. Demerits Not much sufficient for directing the sequencing phase of a genome project. Limited accuracy Depends on the no. of crossovers that have been scored.

15. 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

16. Restriction mapping Restriction mapping locates the relative positions on a DNA molecule of the recognition sequences for restriction endonuclease. 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. Direct examination of DNA molecules for Restriction sites Two ways –gel stretching -molecular combing

17. 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 fibre. In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.

18. 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.

19. Genetic map vs physical map Genetic map Physical map It is constructed using recombination frequency calculated from the progenies It pertains to locating the position of DNA sequences. It is an indirect method of locating the positions of genes or DNA markers It is a direct method of locating the positions of genes or DNA markers. Unit of measurement of map distance is cM Unit of measurement of map distance is base pair.

20. Importance of gene mapping It helps in the analysis of the heterogeneity and segregation of human genetic diseases. It help to develop methods for gene therapy. It provides clinically useful information about linkage

21. Cloning of a disease gene Cloning – a clone is a genetically identical copy of an organism and it ay b naturally occurring or created in the lab. Types of cloning Gene cloning Reproductive cloning Therapeutic cloning

22. Gene cloning Making multiple copies of a single gene The insertion of a fragment of DNA carrying a gene into a cloning vector and subsequent propagation of recombinant DNA molecules into many copies is known as gene cloning

23. Basic steps of gene cloning Construction of recombinant DNA molecule Transport of the recombinant DNA to the host cell Multiplication of recombinant DNA molecule Division of the host cell Numerous cell division resulting in a clone The gene cloning requires specialized tools and techniques Vehicles vector

24. PCR Pcr is a method of copying DNA molecules. DNA replication is common life; for example it takes place inside your own cells every time they divide. PCR, or the polymerase chain reaction, add a two components to this process. The initial reaction yields twice the number of starting molecules, but then is immediately followed by a subsequent reaction. The goal of PCR reaction is commonly to replicate only a portion of the of interest.

25. Uses of PCR It is used in human diagnostics Detecting HIV Detecting bacterial infections Genotyping Environmental monitering Scientific research

26. Three main stages of PCR Denaturing- when the double- stranded template DNA is heated to separate it into two single strands Annealing- when the temperature is lowered to enable the DNA primers to attach to the template DNA Extending- when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

27. References Molecular pharmacology: from DNA to Drug discovery . John dickensonet.al Slide shares Gene mapping Gene cloning

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