DNA SEQUENCING#GK.pptx introduction to sequencing
- 1. DNA SEQUENCING GOKUL KRISHNA T M Roll No 8
- 2. INTRODUCTION • Once a gene or DNA fragment is cloned, studying it further requires DNA sequencing. • DNA sequencing was a very Difficult and time- consuming process before 1975 • In 1975, significant advancements were made in DNA sequencing techniques. • Two different DNA sequencing methods were developed, but only one became widely used - Sanger method And maxam Gilbert method
- 3. MAXAM AND GILBERT'S CHEMICAL DEGRADATION METHOD Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1976–1977. This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides
- 4. In this method, following steps are involved: 1) Label 5' ends of DNA with ³²P 2) Separate two strands, both labelled at 5’ ends. 3) Divide the mixture in four samples, each treated with a different reagent having the property of destroying either only G, or only C, or 'A and G' or 'T and c’. Theconcentration of reagent is so adjusted that 50% of target base is destroyed, so that fragments of different sizes having 32p are produced.
- 5. 4) Electrophorese each of the four Samples in four different lanes of the gel. 5) Autoradiograph the gel and determine the sequence from positions of bands in four lanes.
- 7. 1. FORMIC ACID Breaks the link between a purine (A or G) and the deoxyribose to which it is attached
- 8. 2.DIMETHYL SULFATE Removal of modified G from polynucleotide chain
- 9. 3.HYDRAZINE The pyrimidines (C+T) are hydrolysed using hydrazine
- 10. 4.SODIUM CHLORIDE The addition of sodium chloride to the hydrazine reaction inhibits the reaction of thymine for the only reaction
- 11. 5.PIPERIDINE The modified DNAs cleaved by hot piperidine at the position of the modified base
- 13. SANGER’S DIDEOXY NUCLEOTIDE SYNTHETIC METHOD • Frederick Sanger (who won Nobel Prize twice) had initially developed a method for DNA sequencing (1975) • This method utilized DNA polymerase to extend DNA chain length. • This was termed plus-minus method.
- 14. It was one of the first methods used to determine the order of nucleotides in DNA (1970 s) preceding the more famous chain termination method PRINCIPLE: - The plus-minus method relies on controlled enzymatic synthesis of DNA fragments. It involves two sets of reactions: - PLUS AND MINUS METHOD
- 15. The "plus" reactions, where synthesis includes a specific nucleotide, The "minus" reactions, where synthesis is missing a specific nucleotide. This allows the determination of the sequence by comparing which fragments are present or absent in each reaction.
- 16. DNA Template: The method starts with a single-stranded DNA template that needs to be sequenced Primers: Short DNA primers are annealed to the template to initiate synthesis. DNA Synthesis: DNA polymerase is used to extend the primer, incorporating nucleotides to synthesize a new strand of DNA. PROCEDURE
- 17. Plus and Minus Reactions: The reaction is split into several tubes. In the "plus" reactions, all four nucleotides are present, but in each tube, one nucleotide is marked or emphasized. In the "minus" reactions, one of the four nucleotides is deliberately omitted. This results in a series of fragments where synthesis has terminated at various points, depending on the presence or absence of a specific nucleotide.
- 18. Separation and Analysis: The resulting DNA fragments from each reaction are separated by gel electrophoresis. By comparing the lengths of the fragments in the "plus" and "minus" reactions, the sequence of the DNA template can be inferred.
- 19. The plus-minus method was more complicated and less efficient than later techniques. It required multiple reactions and was labor- intensive, making it difficult to sequence long stretches of DNA accurately LIMITATIONS
- 20. MODIFIED SANGER SEQUENCING METHOD This method was developed by Frederick Sanger in 1975. It was the method used for the ground-breaking Human Genome Project, completed in 2003. Other names for this techniques are 'chain-termination sequencing' or 'dideoxy sequencing'.
- 22. • These dideoxynucleotides are used as triphosphates (ddNTP) and can be incorporated in a growing chain, but they terminate synthesis, • since they cannot form a phosphodiester bond with next incoming deoxynucleotide triphosphate (dNTP).
- 23. Following steps are involved in the Sanger's dideoxy method for DNA sequencing:- (i) Four reaction tubes are set up, each containing single stranded DNA sample (cloned in MI3 phage) to be sequenced, all four dNTPs (radioactively labelled) and an enzyme for DNA synthesis (DNA polymerase l)
- 24. Each tube also contains a small amount (much smaller amount relative to four dNTPs) of one of the four ddNTP, so that four tubes have each a different ddNTP, bringing about termination at a specific base - adenine (A), cytosine (C), guanine (G) and thymine (T) (ii) The fragments, generated by random incorporation of ddNTP leading to termination of reaction, are then separated by electrophoresis on a high resolution Polyacrylamide slab gel.
- 25. This is done for all the four reaction mixtures on adjoining lanes in the gel. (iii) The gel is used for autoradiography so that the Position of different bands in each lane can be visualized. (iv) The bands on autoradiogram can be used for getting the DNA sequence.
- 27. Reference • Elements of Biotechnology. P.K. Gupta • https://www.ncbi.nlm.nih.gov • https://www.britannica.com/science/DNA- sequencing
- 28. Thank you…