Gene mapping and sequencing

Introduction to gene mapping and sequencing . Overview of techniques of gene mapping and sequencing

1. Gene mapping and sequencing Preetam Palkar F.Y. M. Pharmacy AISSMS college of pharmacy

2. Genomics Genomics is the branch of molecular biology concerned with the structure, functions, evolution and mapping of genome and sequencing. The term genomics was coined by Thomas H. Roderick in 1986.

3. Genetic markers A genetic marker is a DNA sequence with a known physical location on a chromosome. They are used to track the inheritance of a nearby gene that has not yet been identified but whose approxi location is known. Example Restriction fragment length polymorphism (RFLP) Simple sequence length polymorphism (SSLP) Single nucleotide polymorphism (SNP)

4. Gene mapping Gene mapping means the mapping of genes to specific locations on chromosomes These maps indicate the locus of genes in the genome and also distance between them. It is very important in case of diagnosis of various genetic diseases.

5. Types of gene mapping Genetic mapping or linkage mapping Relative positions between two genes on a chromosome Physical mapping Precise location of specific DNA sequencing which are to be located .

6. Linkage mapping Gives us an idea whether two genes are linked or not Recombination frequency is measured Recombination frequency = recombinant type/ recombinant type + parental type *100 Example recombinant frequency = 24 /(24+76)*100 = 24% Recombinant frequency is directly proportional to distance between two genes This distance is given in terms of centi Morgan (cM)

7. LOD score LOD stands for "logarithm of the odds.“ In genetics, the LOD score is a statistical estimate of whether two genes, or a gene and a disease gene, are likely to be located near each other on a chromosome and are therefore likely to be inherited. A LOD score of 3 or higher is generally understood to mean that two genes are located close to each other on the chromosome

8. Lod score

9. Physical mapping Also called as Cytogenetic mapping Overlapping of genes is studied to construct a map of whole chromosome Techniques of physical mapping Somatic cell hybridisation Radiation hybridisation Fluorescent in situ hybridisation (FISH)

10. Somatic cell hybridisation

11. Human cell + mouse cells fusion Formation of hybridoma cells having both genes Elimination of either genes Formation of variety of hybrid cell lines. Subculture to obtain more cell line Subjected to southern blotting.

12. Radiation cell hybridisation

13. Human cell + mouse cells (irradiated )fusion Formation of hybridoma cells having both genes Elimination of either genes Formation of variety of hybrid cell lines. Subculture to obtain more cell line Subjected to southern blotting

14. Make a probe complementary to the known sequence. When making the probe, label it with a fluorescent marker, e.g. fluorescein, by incorporating nucleotides that have the marker attached to them. Put the chromosomes on a microscope slide and denature them. Denature the probe and add it to the microscope slide, allowing the probe hybridize to its complementary site. Wash off the excess probe and observe the chromosomes under a fluorescent microscope. The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to.

15. Gene sequencing Gene sequencing is a process in which the individual base nucleotide in an organisms DNA are identified It is the process of determining the precise order of nucleotide within a DNA molecule This sequencing was first proposed by Frederick Sanger in 1975 by chain termination or by dideoxy sequencing Allan maxam and Walter Gilbert developed the DNA sequencing method by chemical modification in 1976

16. Methods of gene sequencing Basic DNA sequencing Sanger sequencing Maxam-Gilbert sequencing Advanced DNA sequencing Shot gun sequencing Next generation DNA sequencing Solid sequencing Illumina sequencing Pyro sequencing

17. Sanger sequencing A DNA primer is attached by hybridization to the template strand and deoxynucleosides triphosphates (dNTPPs) are sequentially added to the primer strand by DNA polymerase. The primer is designed for the known sequences at 3’ end of the template strand. The reaction mixture also contains dideoxy nucleoside triphosphate (ddNTPs) along with usual dNTPs. If during replication ddNTPs is incorporated instead of usual dNTPs in the growing DNA strand then the replication stops at that nucleotide.

18. ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot make phosphodiester bond with nest nucleotide, thus terminates the nucleotide chain Respective ddNTPs of dNTPs terminates chain at their respective site. For example ddATP terminates at A site. Similarly ddCTP, ddGTP and ddTTP terminates at C, G and T site respectively. The reaction mixture from four batches are loaded into four different well on polyacrylamide gel and electrophoresed

19. Maxam-Gilbert sequencing Denature a double-stranded DNA to single-stranded by increasing temperature. Radioactively label one 5' end of the DNA fragment to be sequenced by a kinase reaction using gamma- 32 P. Cleave DNA strand at specific positions using chemical reactions. For example, we can use one of two chemicals followed by piperdine . Dimethyl sulphate selectively attacks purine (A and G), while hydrazine selectively attacks pyrimidines (C and T). Now in four reaction tubes, we will have several differently sized DNA strands. Fragments are electrophoresed in high-resolution acrylamide gels for size separation.

20. Shot gun sequencing The most efficient way to sequence a large piece of DNA involves a process known as shotgun sequencing. For this, the starting DNA is broken up randomly into many smaller pieces . The resulting sequence reads generated from the different pieces are then analyzed by a computer program, looking for stretches of sequence from different reads that are identical with one another. When identical regions are identified, they are overlapped with one another, allowing the two sequence reads to be stitched together. This computer process is repeated over and over and over again, eventually yielding the complete sequence of the starting piece of DNA.

21. Illumina sequencing Illumina dye sequencing  is a technique used to determine the series of base pairs in  DNA , also known as  DNA sequencing . This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It can also be used for whole- genome  and region sequencing,  t ranscriptome  analysis,  meta genomics small  RNA  discovery,  methylation  profiling, and genome-wide  protein nucleic acid interaction analysis.

22. Illumina sequencing technology works in three steps : amplify , sequence and analyze . DNA is fragmented and adapters are added that acts as reference point during amplification ,sequencing and analysis. Modified DNA is loaded onto flow cell where amplification and sequencing takes place . The flowcell contains nanowells that space out fragments and help with overcrowding. Each nanowell contains oligonucleotides that provide an anchoring point for adaptors to attach.

23. Now cluster formation begins . Thousands of copies of each fragment of DNA are formed and it is done by bridge amplification PCR. Now, primers and modified nucleotides are washed onto chip. These nucleotides have reversible 3’ fluorescent blocker so the DNA polymerase can add only one nucleotide at a time. A chemical deblocking step is then used to remove the 3’ terminal blocking group . The process continues until full DNA molecule is sequenced.

24. Advantage Due to automated nature of illumine dye sequencing it is possible to sequence multiple strands at once . Illumina uses only DNA polymerase as opposed to multiple exprnsive enzymes required by other sequencing techniques.

25. Next generation sequencing Ion torrent sequencing Sequencing by oligonucleotide ligation and detect(SOLiD) Pyrosequencing Illumina sequencing Nanopore sequencing Mass spectrophotometric sequencing Direct visualisation of single DNA molecule by atomic force microscopy (AFM) Single nucleotide cutting Readout of cellular gene expression Use of DNA chips or micro arrays

26. Some commercial sequencers Roche1454FLX pyrosequencer – pyrosequcencing Illumina genome analyser- sequencing by synthesis Applied bio system SOLiD sequencer- sequencing by ligation Helicon heliscope Pacific biosciences SMRT – zero ode waveguide

27. Reference National human genome research institute Nachimuthu Saraswathy, Ponnusamy Ramalingam, in Concepts and Techniques in Genomics and Proteomics, 2011   Slatko , B. E., Gardner, A. F., & Ausubel , F. M. (2018). Overview ofnext-generation sequencing technologies. Current Protocols in Molecular Biology, 122, e59. doi : 10.1002/ cpmb.59 Kelly L. Williams, in Encyclopedia of Bioinformatics and Computational Biology, 2019 M.F. Seldin , in Encyclopedia of Genetics, 2001