DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING

This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis

1. DNA SEQUENCING METHODS & STRATEGIES FOR GENOME SEQUENCING Presented By:- Neeraj Chaturvedi Nikhat Fatima Kaynat Arif Kirtika Adhikari Puneet Kulyana METHODS FOR ANALYSIS OF GENE EXPRESSION AT RNA AND PROTEIN LEVEL & LARGE SCALE EXPRESSION ANALYSIS 1

2. DNA SEQUENCING DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—Adenine ,  Guanine ,  Cytosine , and  Thymine—in a strand of DNA 2

3. METHODS OF DNA SEQUENCING Basic methods Maxam -Gilbert sequencing Chain-termination methods Advanced methods and  de novo  sequencing Shotgun sequencing Bridge PCR High-throughput methods 3

4. Maxam –Gilbert sequencing Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. It was the first widely adopted method for DNA sequencing, and, along with the Sanger dideoxy method, represents the first generation of DNA sequencing methods. 4

5. PROCEDURE:- Radioactive labeling  is done at one 5′ end of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions . The modified DNAs may then be cleaved . Thus a series of labeled fragments is generated, which are then  electrophoresed  side by side in denaturing acrylamide gels for size separation To visualize the fragments, the gel is exposed to X-ray film  5

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7. CHAIN TERMINATION METHOD Sanger’s method is also known as dideoxy chain termination method. It was developed by the British biochemist Fred Sanger and his colleagues in 1977. PRINCIPLE: The Sanger Technique uses dideoxynucleotides ( dideoxyadenine , dideoxyguanine , etc) These are molecules that resemble normal nucleotides but lack the normal -OH group. Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops. 7

8. Procedure:- 1. Template preparation:- Copies of template strand to be sequenced must be prepared with short known sequences at 3’ end of the template strand. 2. Generation of nested set of labelled fragments:- Copies of each template is divided into four batches and each batch is used for different replication reaction. To synthesize fragments that terminates at A, ddATP is added to the reaction mixture on batch I along with dATP , dTTP , dCTP and dGTP , standard primer and DNA polymerase I. 8

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10. 3. Electrophoresis:- The reaction mixture from four batches are loaded into four different well on polyacrylamide gel and electrophoresed. The autoradiogram of the gel is read to determine the order of bases of complementary strand to that of template strand. 4. Gel Separation :- The reaction mixtures are separated on a denaturing polyacrylamide gel. Polyacrylamide can separate the DNA strands which differ in length by only one nucleotide. The separated neucleotides are separated by radioactive method or fluorescent method. 10

11. Applications Molecular biology:- To study genomes and the proteins they encode. Evolutionary biology:- Is used in  evolutionary biology to study how different organisms are related and how they evolved. Metagenomics:- It involves identification of organisms present in a body of water,  sewage , dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research. Medicine:- Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. Forensics:- DNA sequencing may be used along with  DNA profiling  methods for  forensic identification [4]  and  paternity testing . 11

12. What is a genome??? Term “genome” was coined by H. Winkler in 1920 A genome can be defined as the entire DNA content of each nucleated cell in an organism Humans for example have a genome that is encoded on 46 chromosomes, organized into 23 pairs, out of which 44 chromosomes are autosomes and 2 are sex chromosomes Each genome contains all of the information needed to build that organism and allow it to grow and develop Genome is a unique sequence of DNA It is over 3 billion letters long and is found in almost every cell in the body 12

13. What is Genome sequencing??? Genome sequencing is the technique that allows researchers to read the genetic information found in the DNA of anything from bacteria to plants to animals Sequencing involves determining the order of bases, the nucleotide subunits- Adenine(A), Guanine(G), Cytosine(C) and Thymine(T), found in DNA It has largely been used as a research tool, but is currently being introduces to clinics Genome sequencing should not be confused with DNA profiling, which only determines the likelihood that genetic material came from a particular individual or group and doesn’t contain information on origin or susceptibility to specific diseases Sequencing a genome is an enormous task. It requires not only finding the nucleotide sequence of small pieces of genome but also ordering those small pieces together into the whole genome. 13

14. Steps of genome sequencing Break genome into smaller fragments Sequence those smaller pieces Place the sequences of the short fragments together 14

15. Genome sequencing Two different methods used :- Hierarchical shotgun sequencing – useful for sequencing genomes of higher vertebrates that contain repetitive sequences Whole genome shotgun sequencing – useful for smaller genome Factors that determine sequencing strategy are :- Genome size Chromosomal structure Repeat content and character Desired end product 15

16. Hierarchical shotgun sequencing The method preferred by the HGP is the hierarchical shotgun sequencing method. Also known as - The Clone-by-clone strategy - The map-based method - map first, sequence later - top-down sequencing In clone based sequencing the first step is mapping. One first constructs a map of the chromosome, making them at regular intervals of about 100 kilo bases. Then known segments of marked chromosome are cloned in plasmids. One special type of plasmid used for genome sequencing is BAC, which contain DNA fragment of about 80-180 kb in E.Coli cells. The plasmid cells are then further broken into small, random, overlapping fragments of 0.5 to 1.0 kb. Finally automated sequencing machines determine the order of each nucleotide of the small fragments. The National Human Genome Research Institute used clone based sequencing for the human genome, for this they relied on computer scientists to assemble the final sequence 16

17. Whole genome sequencing by shotgun J. Craig Venter and H. Smith developed “whole genome shotgun” sequencing and sequenced the genome of bacteria H. Influenza and M. genitalicum . This approach maybe characterized into 4 steps :- Library construction : the chromosome is isolated from the desired cells following the methods of molecular biology and randomly fragments into small pieces using ultrasonic waves. Then the fragments are purified and attached to the plasmid vector. Plasmids with single insert are isolated. A library of plasmid clone is prepared transforming E. Coli strains with plasmid lacked restriction enzymes. Random sequencing : the DNA is purified from the plasmid. Thousands of DNA fragments are sequenced using automated sequencer. Fragment alignment and gap closure : by using special computer programmes , the sequenced DNA fragments are clustered. Two fragments are joined together to form large stretch of DNA Proof reading : proof reading is done carefully so that any ambiguities could be resolved. The sequence is also checked for frameshift mutation if so, the mutation is corrected. 17

18. Hybrid shotgun – sequencing strategy In this, sequence reads are generated in both a clone-by-clone and a whole genome fashion. A hybrid shotgun- sequencing strategy can, in principle, capture the advantageous elements of both clone-by-clone and whole-genome approaches. For eg . The whole-genome shotgun component provides the rapid insight about the sequence of the entire genome. 18

19. Next generation sequencing Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies including: Illumina ( Solexa ) sequencing Roche 454 sequencing Ion torrent: Proton / PGM sequencing SOLiD sequencing These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such have revolutionised the study of genomics and molecular biology. 19

20. aDVAntages & Disadvantages of Genome sequencing ADVANTAGES DISADVANTAGES Normalized coverage of all genes Information about gene structure Information about regulatory elements Genome organization High cost Time consuming Difficult to determine if a sequence codes for a gene 20

21. METHODS FOR ANALYSIS OF GENE EXPRESSION AT RNA AND PROTEIN 21

22. GENE EXPRESSION Gene expression is a highly regulated mechanism that controls the function and adaptability of all living cells including prokaryotes and eukaryotes. Several techniques exist for studying and quantifying gene expression and its regulation. Some of these techniques are old and well established while others are relatively new, multiplex techniques. The field of gene expression analysis has undergone major advances in biomedical research. Traditional methods focused on measuring the expression of one gene at a time and not in any particular biological context. However , today, mRNA expression techniques have led to improvements in gene identification and disease sub-classification, for example. 22

23. METHODS OF GENE EXPRESSION  AT RNA AND PROTEIN level SAGE Electrophoresis Microarray Western Blotting Proteomics Immunohistochemistry 23

24. SAGE: Based on 2 principles SAGE is a technique used to create a library of short sequence tags which can each be used to detect a transcript. The expression level of the transcript can be determined by assessing how many times each tag is detected. This technology enables comprehensive expression analysis across the genome. A 9 bp tag is sufficient to unambiguously identify a gene Concatenation (linking together) of these short DNA sequences increases the efficiency of identifying unique transcripts in a serial manner.  24

25. Methods 1. mRNA   to cDNA 2 . Cleave with A.E 3 . Isolate 3’ most transcript of each cDNA by binding to Streptavidin beads 4 . Divide cDNA in half 5 . Ligate to 1 of 2 linkers ( each with a T.E site ) 6 . Ligate the two pools of tags together…. 25

26. 7 . Ligated linkers serve as primers for amplification 8 . Cleave PCR products with A.E. to isolate ditags 9 . Concatenate by ligation 10 . Clone 11 . Sequence 26

27. Current Applications Gene Discovery Analysis of Cardiovascular gene expression Gene expression in carcinogenesis Substance abuse studies Cell , tissue and developmental stage profiles in C. elegans Profiling of human diseases and more….. 27

28. WESTERN BLOTTING  A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. So called since it has some similarity to a Southern blot. The Western Blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Western blotting is a technique for detecting specific protein molecules within a protein mixture. This mixture might include all the proteins that are associated with a certain cell type or tissue. The technique can help to determine a protein’s size, and how much of it is expressed. 28

29. Advantages of Western Blot    Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. 29

30. The procedure includes.. Tissue preparation  Gel electrophoresis Transfer   Blocking   Detection Analysis   30

31.  Tissue Preparations  Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down  mechanically using a blender. It should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Tissue preparation is often done at cold temperatures to avoid protein denaturing.  31

32. Gel Electrophoresis The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point molecular weight, electric charge, or a combination of these factors. The principle involved is the difference in the ELECTROPHORETIC MOBILITIES of different proteins.  32

33. Transferring  In order to make the proteins accessible to antibody  detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF ). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.  33

34. Blocking The membrane has the ability to bind to proteins in in this case both the target and antibodies are proteins and so there could be some unwanted binding. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin(BSA) with a minute percentage of detergent such as Tween 20. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. Blocking Presented by Pri 34

35. Detection  During the detection process, the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colorimetric reaction and produces a color.  35

36. Analysis   After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest .  Size approximations are taken by comparing the stained bands to that of the marker loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin that should not change between samples.  36

37. Advantages While ELISA being a non specific test, Western blotting is a more specific test for detection of HIV. It can detect one protein in a mixture of proteins while giving information about the size of the protein and so is more specific. Western blot test is referred to as the 'Gold Standard’ It also tells you how much protein has accumulated in cells . 37 DISADVANTAGES  If a protein is degraded quickly, Western blotting won't detect it well  This test takes longer that other existing tests It might also be more costly

38. gene expression analysis Functional genomics involves the analysis of large datasets of information derived from various biological experiments. One such type of large-scale experiment involves monitoring the expression levels of thousands of genes simultaneously under a particular condition , called gene expression analysis. Microarray technology makes this possible and the quantity of data generated from each experiment is enormous, dwarfing the amount of data generated by genome sequencing projects . All of our cells carry some amount and type of genes Expression varies from type of cells(normal vs tumour ), from time to time (different development stages), & environmental response Microarray is used to detect DNA, protein, RNA, antibodies etc. Most commonly used DNA microarray 38

39. DNA Microarray( dna chip) It measures amount of mRNA in a given cell at a particular time It is divided into 2 stages: STAGE 1 :- produce DNA chip: - extract DNA - amplify using PCR - DNA denaturation and get ssDNA STAGE 2 :- transcriptome: - total mRNA extract at a particular time from a particular cell - reverse transcribe and produce cDNA using reverse transcriptase This cDNA works as control and tagged with cy3 dye(green) 39

40. Applications of DnA Microarray There are various applications of DNA microarray. Some of them are:- To check which gene is expressed in a particular cell and condition To evaluate which therapy is best Which specific genes are affected in a variety of disease (heart disease, diabetes, cancer etc.) Classification of disease 40

41. REFERENCES https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831009/ www.ebi.ac.uk/.../what-next-generation- dna - https://www.ncbi.nlm.nih.gov/pubmed/20486139http:// www.cell.com/molecular-cell/abstract/S1097-2765(15)00340-8 www.google.co.in 41

42. THANKYOU 42