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Dna sequencing

The document discusses DNA sequencing methods. It describes two early conventional methods: Sanger chain termination sequencing and Maxam-Gilbert chemical degradation. Sanger's method uses dideoxynucleotides as chain terminators and is more efficient with fewer toxic chemicals. The document also explains pyrosequencing, an early next-generation sequencing technique that detects pyrophosphate release upon nucleotide incorporation.

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DNA SEQUENCING
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
To determine the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used 1. Sanger; Chain Termination Sequencing method 2. Maxam and Gilbert; Chemical Sequencing method These two methods are most popular conventional methods Robotics and automated sequencing are based on these methods
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
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
Chain termination method of DNA sequencing • It involves following components: 1. Primer 2. DNA template 3.DNA polymerase 4.dNTPs(A,T,G,C) 5. ddNTPs • It involves following 4 Steps: 1. Denaturation 2. Primer attachment and extension of bases 3. Termination 4. Poly acrylamide gel electrophoresis
• The Methods (Procedure ):- 1. Before the DNA can be sequenced, it has to be denatured into single strands using heat. 2. 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".
• ‘’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
• 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
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
• 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
• 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.
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.
Maxam Gilbert Sequencing: Process Summarized 1. Label 5’- end of DNA 2. Aliquot DNA sample in 4 tubes 3. Perform base modification reaction 4. Perform Cleavage reaction 5. Perform Gel Electrophoresis 6. Perform Autoradiography 7. Interpret results
I. Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP) II. Purification of the DNA fragment to be sequenced III. Chemical treatment generates breaks in DNA IV.Run on the gel
Chemical Modification and Cleavage Poly nucleotide Kinase radioactive label at one 5' end of the DNA using gamma-32P 5′ G A C G T G C A A C G A A 3′ 32P 5′ G A C G T G C A A C G A A 3′
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
Base modification using Hydrazine – Pyrimidine • Cytosine • Thymine – Hydrazine----- C+T – Hydrazine + NaCl--------C Cleavage of Sugar Phosphate backbone using Piperidine
An Example for Maxam-Gilbert Sequencing
1. 454 Pyrosequencing Pyrosequencing is based on the 'sequencing by synthesis' principle, where a complementary strand is synthesized in the presence of polymerase enzyme.
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
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).
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.