DNA SEQUENCING

DNA SEQUENCING

It is the process of determining the precise order of nucleotide within a DNA molecule.

First DNA sequencing is obtained in the early 1970,by academic researches usinglaborious method based two dimensional chromatography.

Four canonical bases : • Adenine• Guanine• Thymine• Cytosine

History• 1953 - structure of DNA established as a double helix.

• RNA sequencing was one of the earliest forms of nucleotide sequencing.

• 1970 - first method of DNA sequencing involved a location specific primer extension strategy.

• 1977 - Frederick sanger published a method for DNA sequencing with chain terminating inhibitors.

• 1977 - Allan Maxam and Walter Gilbert developed DNA sequencing by chemical degradation.

• 1977 - the first genome to be sequenced was that of bacteriophage φX174.

• 1990 - several new methods are developed in the mid to late 90’s.

• 2003 - Complete Human Genome Project.

Methods of DNA sequencing Basic methods: 1. Maxam-Gilbert sequencing

2. Chain termination method

Advanced methods: 1.Short Gun Sequencing 2.Bridge PCR Sequencing

Next generation methods: 1. Massively parallel signature sequencing 2. Polony sequence 3. Pyrosequencing 4. Illumina sequencing 5. Solid sequencing 6. DNA nanoball sequencing.

Maxam And Gilbert Sequencing:

10 nucleotide DNA sequence: 5’P-TTCAGCCGAT-OH3’ First step: 5’P-TTCAGCCGAT-OH3’+H2O 5’OH-TTCAGCCGAT-OH3’+Pi

5’OH-TTCAGCCGAT-OH3’+A-P-P-32P 5’32P-TTCAGCCGAT-OH3’+ADP [gamma-32P]ATP

The DNA solution is divided into four aliquots: 1. G only 2. G+A 3. C+T 4. C only G only G+A

C+T C only

1. G only : - In this tube the DNA is incubated with dimethyl sulfate (DMS). - 5’32P-TTCA and 5’32P-TTCAGCC- two G residue present. - one strand will be 4 nucleotide other will be 7 nucleotide long.

2. A+G : - Here the DNA is protonated rather than methylated. - TTC, TTCA, TTCAGCC, TTCAGCCG. - measuring 3,4 ,7 and 8 nucleotides in length.

3. C+T : - DNA is reacted with hydrazine(NH2-NH2) and this followed with piperidine treatment. - T, TT, TTCAG, TTCAGC, TTCAGCCGA. - measuring 1, 2, 5, 6 and 9 nucleotide in length

Next steps:

- The four differently fragmented sample of DNA are simultaneously electrophoresed in parallel lanes on a sequencing gel

- After electrophoresis gel is exposed to a photographic film

- The sequence of DNA simply read of f this autoradiogram

4. C only :

- If hydrazine treatment is carried out in presence of 1.5M NaCl. - TT, TTCAG, TTCAGC. - measuring 2, 5,and 6 nucleotides long.

SUMMARY………

THE SANGER CHAIN - TERMINATION METHOD•Developed in MID-1970 by TWO scientists SANGER and

A.R.COULSON

• It is an ENZYMATIC method

• PRINCIPLE:

•Use of DIDEOXY NUCLEOSIDE TRIPHOSPHATES (ddNTP) as DNA Chain terminators

REQUIRMENTS:• ssDNA Fragment

•DNA Primer

•DNA Polymerase

•All FOUR DEOXY RIBONUCLEOSIDE TRIPHOSPHATES

•Small con. Of the DI-DEOXY NUCLEOSIDES TRI-PHOSPHATES or ddNTP

Ex: dd ATP

dd GTP

dd CTP & dd TTP

Difference between d NTP & dd NTPs:

• A ddNTP is a laboratory made chemical molecule which is act as a ANALOGUE to dNTP

• Its LACKS the HYDROXYL group at both the 2’ and 3’ carbons of the sugar

• Significance of 3’ hydroxyl group– Formation of phosphodiester bond

•DNA SEQUENCING IS CARRIED OUT IN FOUR REACTION TUBES IN 4 STEPS

•STEP 1: DENATURATION

•STEP 2: PRIMER ATTACHMENT AND EXTENSION OF BASES

•STEP 3: TERMINATION

•STEP 4: POLY ACRYLAMIDE GEL ELECTROPHORESIS

Sanger Method Automated fluorescent DNA sequencing method

Primer is radio labelled with either 32p or 35s

ddNTP labelled with 4 different FLUORESCENT dyes

There is only a single round of DNA synthesis

Hybridization, Synthesis and Denaturation is repeated many times

Sequences are carried out in 4 reaction tubes

Four chain-terminated products are run on the same tube

300bp 500bp to 100,000bp

PYRO SEQUENCING

•DNA Sequencing based on the “SEQUENCING BY SYNTHESIS”

•Its relies on the detection of PYROPHOSPHATE release on NUCLEOTIDE incorporation, rather than CHAIN TERMINATION

•The single-strand DNA template is hybridized to a sequencing primer and incubated with the enzymes

•The pyrosequencing method is based on detecting the activity of DNA polymerase  with another chemiluminescent enzyme

•DNA polymerase

• ATP sulfurylase

•Luciferase and apyrase

•Substrates adenosine 5´ phosphosulfate (APS) and luciferin

APPLICATIONS OF DNA SEQUENCING:• To Find Genes

• Information regarding MUTATIONS

• Gene overlapping

• Identification of POLYMORPHSIMS

• Profiling of the DNA methylation in the genomes

• Exome sequencing

• Identification of GENE REGULATORY CONTROL SEQUENCES

• To determine the PATERNITY of a child

REFERENCES:• TEXT BOOK OF BIOCHEMISTRY BY D.M.VASUDEVAN

• BIO PHYSICAL CHEMISTRY BY UPADHYAYA AND NATH

• TIETZ TEXTBOOK OF CLINICAL CHEMISTRY

• MOLECULAR BIOLOGY— BURTAN .E. TROPP

• WILSON AND WALKER PRINCIPLES AND TECHNIQUES OF BIOCHEMISTRY 

• CELL AND MOLECULAR BIOLOGY– E.D.P.DE ROBERTI'S 8TH EDITION

• BIOINFORMATICS– PRACTICAL APPROACH– SHUI QING YE

• TEXTBOOK OF BIOCHEMISTRY BY U SATYANARAYANA

• HTTP://EN.WIKIPEDIA.ORG/WIKI/DNA_SEQUENCING --PYRO SEQUENCING

• DNA SEQUENCING WRITTEN BY: ANTHONY J.F. GRIFFITHS

THANK YOU…..