Dna sequencing

DNA Sequencing and its methods

1. DNA SEQUENCING

2. What Is DNA Sequencing ? The term DNA Sequencing refers to method for determining the order of the nucleotide bases Adenine, Guanine, Cytosine and Thymine in a molecule of DNA

3. To determine the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used Sanger; Chain Termination Sequencing method Maxam and Gilbert; Chemical Sequencing method These two methods are most popular conventional methods Robotics and automated sequencing are based on these methods

4. Disadvantages Of Maxam & Gilbert Chemical Degradation Method Requires lots of purified DNA and many intermediate purification step It requires extensive use of hazardous chemicals It is difficult to scale up and cannot be used to analyze more than 500bp

5. SANGERS METHOD OF DNA SEQUENCING The chain terminator method is more efficient and uses fewer toxic chemicals and lower amount of radioactivity than the method of Maxam and Gilbert. The key principle of the Sangers method was use of dideoxynucleotides triphosphate (ddNTPs) as DNA chain terminator

6. Chain termination method of DNA sequencing It involves following components: Primer DNA template DNA polymerase 4.dNTPs(A,T,G,C) 5. ddNTPs It involves following 4 Steps: Denaturation Primer attachment and extension of bases Termination Poly acrylamide gel electrophoresis

7. The Methods (Procedure ):- Before the DNA can be sequenced, it has to be denatured into single strands using heat. Next a primer is annealed to one of the template strands. This primer is specifically constructed so that its 3' end is located next to the DNA sequence of interest. Either this primer or one of the nucleotides should be radioactively or fluorescently labeled so that the final product can be detected on a gel. Once the primer is attached to the DNA, the solution is divided into four tubes labeled "G", "A", "T" and "C".

8. ‘’G’’ tubes : all four dNTP’s, ddGTP and DNA polymerase ‘’A’’ tubes : all four dNTP’s, ddATP and DNA polymerase ‘’T’’ tubes : all four dNTP’s, ddTTP and DNA polymerase ‘’C’’ tubes : all four dNTP’s, ddCTP and DNA polymerase

9. Mixture first heated so that DNA strands separate (96⁰C) Then temperature get lower so that short length DNA sequence – a primer can bind to the template DNA(50⁰C) Temperature raised to (60⁰C- 65⁰C)to enable the DNA Polymerase enzyme to bind to the short section of double stranded DNA. DNA polymerase to synthesizes new DNA. DNA Polymerase will continue adding Nucleotides to chain until it happens to add a dideoxy nucleotide instead of normal one

10. Uses of Sangers sequence Region up to about 900 basepairs in length sequenced using this method In Human Genome project sanger sequencing was used to determine the sequence of relatively small fragments of human DNA Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning

11. Advantage of Basic method 1:-Improvement diagnosis of disease. 2:- Identifying suspects . Disadvantage : 1:-Whole genome can not be sequenced at once . 2:- Very slow and time consuming

12. Applications of DNA Sequencing Forensics: to help identify individuals because each individual has a different genetic sequence Medicine: can be used to help detect the genes which are linked to various genetic disorders such as muscular dystrophy. Agriculture: The mapping and sequencing of a genome of microorganisms has helped to make them useful for crops and food plants.

13. 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.

14. Maxam Gilbert Sequencing: Process Summarized Label 5’- end of DNA Aliquot DNA sample in 4 tubes Perform base modification reaction Perform Cleavage reaction Perform Gel Electrophoresis Perform Autoradiography Interpret results

15. I. Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma- 32 P ATP) Purification of the DNA fragment to be sequenced Chemical treatment generates breaks in DNA Run on the gel

16. Chemical Modification and Cleavage Poly nucleotide Kinase radioactive label at one 5' end of the DNA using gamma- 32 P 5′ G A C G T G C A A C G A A 3′ 32 P 5′ G A C G T G C A A C G A A 3′

17. Chemical Modification and Cleavage Base Modification using Dimethyl sulphate Purine Adenine Guanine Only DMS------- G DMS+ Formic acid-------G+A Cleavage of Sugar Phosphate backbone using Piperidine

18. Base modification using Hydrazine Pyrimidine Cytosine Thymine Hydrazine----- C+T Hydrazine + NaCl--------C Cleavage of Sugar Phosphate backbone using Piperidine

19. An Example for Maxam-Gilbert Sequencing

20. 1. 454 Pyrosequencing Pyrosequencing is based on the 'sequencing by synthesis' principle, where a complementary strand is synthesized in the presence of polymerase enzyme.

21. It initially uses the emulsion PCR technique to construct the colonies required for sequencing and removes the complementary strand. Next, a ssDNA sequencing primer hybridizes to the end of the strand (primer-binding region), then the four different dNTPs are then sequentially made to flow in and out of the wells over the colonies. When the correct dNTP is enzymatically incorporated into the strand, it causes release of pyrophosphate

22. In the presence of ATP sulfurylase and adenosine, the pyrophosphate is converted into ATP. This ATP molecule is used for luciferase-catalysed conversion of luciferin to oxyluciferin, which produces light that can be detected with a camera. The relative intensity of light is proportional to the amount of base added (i.e. a peak of twice the intensity indicates two identical bases have been added in succession).

23. Pyrosequencing, developed by 454 Life Sciences, was one of the early successes of Next-generation sequencing; indeed, 454 Life Sciences produced the first commercially available Next-generation sequencer. However, the method was eclipsed by other technologies and, in 2013, new owners Roche announced the closure of 454 Life Sciences and the discontinuation of the 454 pyrosequencing platform.