![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](https://image.slidesharecdn.com/dnasequencing-150420102620-conversion-gate02/75/DNA-sequencing-7-2048.jpg)
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DNA sequencing
- 2. DNA SEQUENCING It isthe process of determining the precise order of nucleotide within a DNA molecule. First DNA sequencing is obtained in the early 1970,by academic researches using laborious method based two dimensional chromatography. Four canonical bases : • Adenine • Guanine • Thymine • Cytosine
- 3. 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.
- 4. • 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.
- 5. Methods of DNAsequencing Basic methods: 1. Maxam-Gilbert sequencing 2. Chain termination method Advanced methods: 1.Short Gun Sequencing 2.Bridge PCR Sequencing
- 6. Next generation methods: 1.Massively parallel signature sequencing 2. Polony sequence 3. Pyrosequencing 4. Illumina sequencing 5. Solid sequencing 6. DNA nanoball sequencing.
- 7. Maxam And GilbertSequencing: 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
- 8. 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
- 9. Next steps: - Thefour 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.
- 10.
- 11. 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
- 12. REQUIRMENTS: •ssDNA Fragment •DNA Primer •DNAPolymerase •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
- 13. Difference between dNTP & 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
- 14. •DNA SEQUENCING ISCARRIED 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
- 16. Sanger Method Automatedfluorescent 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
- 17. PYRO SEQUENCING •DNA Sequencingbased on the “SEQUENCING BY SYNTHESIS” •Its relies on the detection of PYROPHOSPHATE release on NUCLEOTIDE incorporation, rather than CHAIN TERMINATION
- 18. •The single-strand DNAtemplate 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
- 20. APPLICATIONS OF DNASEQUENCING: • 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
- 21. REFERENCES: • TEXT BOOKOF 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
Editor's Notes
- #8 -Radioactively label one end of the single stranded DNA. Incubated with []ATP and the enzyme polynucleotide kinase from E.coli infected with bacteriophage T4.
- #14 DNA polymerase connects adjacent deoxyribonucleotides by catalyzing a covalent bond between the 5ʹ phosphate on one nucleotide and the 3ʹ –OH group on the previous nucleotide
- #21 The exome represents less than 2% of the human genome, but contains ~85% of known disease-causing variants With exome sequencing, you can investigate the protein coding regions of the genome when sequencing an entire genome is not practical or necessary. It can efficiently identify variants across a wide range of applications, including population genetics, genetic disease, and cancer studies.b