I.P COLLEGE,CAMPUS II

A Presentation on topic

DNA Sequencing

Submitted by-

Meenu sharma

M.Sc. biotechnology (II SEM)

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WHAT IS DNA ?A.DNA stands for Deoxyribo nucleicacids.

B. DNA is a molecule that carries mostof the genetic instructions used in thegrowth, development, functioning andreproduction of all known livingorganisms and many viruses.

C.DNA molecule consist of biopolymerstrands coiled around each other toform a double helix.

D.The two strands are known aspolynucleotide strands since theyconsist of simpler units called called anucleotides.

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STRUCTURE OF A

DNA NUCLEOTIDE-

A. Each nucleotide

consist of a nitrogen

containing nucleo

base either cytosine

(c),guanine (G)

,adenine (A), or

thymine (T) as well as

sugar called deoxy

ribose and a phosphate

group..

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WHAT IS DNA SEQUENCING ?

• The process of determining the order of bases adenine (A), thymine (T), cytosine (C), and guanine (G) along a DNA strand.

• All the information required for the growth and development of an organism is encoded in the DNA of its genome.

• So, DNA sequencing is fundamental to genome analysis and

understanding the biological processes in general.

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METHODS OF DNA SEQUENCING –

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METHODS

A.MAXAM GILBERT

CHEMICAL CLEAVAGE METHOD

B.SANGER DIDEOXY (PRIMER

EXTENSION /CHAIN

TERMINATION METHOD.

C.AUTOMATED DNA

SEQUENCING METHOD.

A. CHEMICAL CLEAVAGE METHOD BY

ALLAN MAXAM AND WALTER GILBERT

(1976-1977)-

• Sequences DNA fragments containing upto ~500 nucleotides in

length.

• This method uses double-stranded DNA samples.

• Maxam -Gilbert sequencing was the first widely used method for

DNA sequencing.

• Involves modification of the bases in DNA followed by chemical

base-specific cleavage.

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STAGES-1. The double-stranded fragment to be sequenced is isolated and

radioactively labeled at the 5’-ends with 32P.

2. The fragment is then cut with restriction enzyme and thus the label is removed from one end.

3. The fragment of DNA with one end labeled is denatured.

4. Four identical samples of these end-labeled DNA restriction fragments are subjected to chemical cleavage at different chemical nucleotides.

5.There are four specific sets of chemical reactions that selectively cut the DNA backbone at G, A+G, C+T, or C residues.

– G only: Dimethyl sulphate(DMS)

and piperidine

– A+G : DMS, piperidine

– C+T : Hydrazine, piperidine

– C only : Hydrazine, alkali,

piperidine7

6. For each labeled chain to be broken only once, the reactions are controlled. 7. The labeled sub-fragments created by the four reactions have the 32P label at one end and the chemical cleavage point at the other end.

8. The reaction products are separated by poly acryl amide gel

electrophoresis which h is based on size. Smallest fragment goes

fastest

9. The labeled fragments in the gel are visualized by

autoradiography.

10. The sequence is read from bottom to top of the gel.

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B.The Enzymatic method /Sanger dideoxy

(Primer extension/chain termination

method)

• The enzymatic procedure is commonly refered to as sanger-

coulson method since it was developed by F.sanger and

coworkers.

• Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain terminating dideoxynucleotides by DNA polymerase during invitro DNA Replication.

• The classical chain- termination method requires –

• 1.A single stranded DNA template.

• 2.A DNA polymerase.

• 3.Normal dideoxynucleotide triphosphates (ddNTP’s).10

STAGES-1.The DNA to be sequenced is called the template DNA. It is prepared as a single-stranded DNA after being spliced into M13 vector DNA. Infected E. coli host cells release phage particles which contains single-stranded recombinant DNA that includes the sample DNA. This DNA sample is then extracted from phage for sequencing purpose. 2. A synthetic 5’-end-labeled oligo deoxynucleotide is used as the primer. 3. The template DNA is hybridized to the primer.4. The primer elongation is performed in four separate polymerization reaction mixtures. Each mixture contains

- 4 normal deoxynucleotides (dNTPs) in higher concentration and

- a low concentration of the each of the 4 ddNTPs.

5.There is initiation of DNA synthesis by adding enzyme DNA polymeras since the enzyme cannot distinguish between the normal nucleotides and their analogues.

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6. The strand synthesis continues until a ddNTP is added. The chain elongation ceases on the incorporation of a ddNTP because it lacks a 3’-OH group which prevents addition of the next nucleotide.7. There is a result of mixture of terminated fragments, all of different lengths. 8. Denature DNA fragments.

9. Each of the four mixtures are run together on a polyacrylamide

gel for electrphoresis.

10. The separated fragments are then visualized by

autoradiography.

11. From the position of the bands of the resulting autoradiogram,

the sequence of the original DNA template strand can be read

directly

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SANGER DIDEOXY CHAIN TERMINATION METHOD

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APPLICATIONS OF DNA

SEQUENCING-

• DNA sequencing may be used to determine thesequence of individual genes , larger genetic regions(i.e, cluster of genes or operons ),full chromosome orentire genomes.

•IN FORENSIC SCIENCE-DNA Sequencing hasbeen used in forensic science-to identify particularindividual because every individual has uniquesequence of his/ her DNA.

-to identify criminals by finding some proof from thecrime scene in the form of hair,nail,skin or bloodsamples.

-to determine paternity of the child .

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•IN MEDICAL RESEARCH-a.DNA sequencing can

be used to detect the genes which are associated with

some heredity or acquired diseases.

-scientists use different techniques like gene therapy

to identify the defected gene and replace them with

the healthy one.

•IN AGRICULTURE- DNA sequencing has played a

vital role in the field of agriculture. The mapping and

sequencing of the whole genome of microorganisms

has allowed the agriculturists to make them useful for

the crops and food plants.

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