DNA sequencing
Transcript of DNA sequencing
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DNA SEQUENCING
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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
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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.
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• 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.
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Methods of DNA sequencing Basic methods: 1. Maxam-Gilbert sequencing
2. Chain termination method
Advanced methods: 1.Short Gun Sequencing 2.Bridge PCR Sequencing
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Next generation methods: 1. Massively parallel signature sequencing 2. Polony sequence 3. Pyrosequencing 4. Illumina sequencing 5. Solid sequencing 6. DNA nanoball sequencing.
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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
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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
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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.
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SUMMARY………
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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
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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
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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
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•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
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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
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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
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•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
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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
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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
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THANK YOU…..