Maxam & Gilbert sequencing

Transcription

1 enomics

2 Beginnings he first protein sequenced was bovine insulin in his was basically done by a series of tricks. Each individual amino acid was determined by a separate and different experiment.

3 Beginnings he first protein sequenced was bovine insulin in his was basically done by a series of tricks. Each individual amino acid was determined by a separate and different experiment. he first direct attempts to sequence an RN molecule were by Holley and co-workers in 1965 (R.W. Holley et al., 1965, Science 147: ). he technique that they used was very labor intensive and it took them approximately one year to determine the 77 nucleotides that make up the alanine transfer RN of yeast.

4 enome Sequencing 1956 First protein sequence (Bovine insulin) 1965 Holley et al. Sience 147: (yeast alanine N) 1977 Maxam and ilbert. NS 74: Sanger et al. NS 74: First sequenced genome: Haemophilus influenzae 1997 Escherichia. coli and Saccharomyces cerevisae 1998 aenorhabditis elegans genome 2000 Drosophila melanogaster genome 2001 Human genome sequence introduce next generation sequencing (NS)

5 Maxam & ilbert sequencing rinciple radioactively label DN fragments at their 5 end using alkaline phosphatase / polynucleotide kinase separate the fragments according to their size using gel electrophoresis followed by an autoradiography to visualize the fragments Method: Four different treatments Dimethylsulfate followed by heat treatment () + mild acid (+) Hydrazine (+) Hydrazine + 2M Nal () Limitation Require a cloning step (amplification and labeling)

6 Maxam & ilbert sequencing ve +ve Inferred DN sequence + +

7 Sanger sequencing rinciple Use the properties of DN replication herefore requires the use of primers im: amplification of fragments of different sizes by stopping the DN replication with the use of 2,3 - dideoxyribonucleotide triphosphates Method: Four different individual reactions are performed with each of the radioactively labeled 2,3 -dideoxyribonucleotide triphosphates el electrophoresis followed by autoradiography

8 Sanger sequencing H2 H2 H H2 H H2 H2 H2 H

9 Sanger sequencing H 2

10 Sanger sequencing DN Replication - H2 - H2 - H2 - H2 H H2 - H2 H H

11 Sanger sequencing - H2 - H2 H H2 H

12 Sanger sequencing - H2 - H2 H H2 dideoxynucleotide triphosphate

13 Sanger sequencing - H2 - H2 - H2 - H2 H H2 - H2

14 Sanger sequencing ve +ve Inferred DN sequence Dideoxynucleotide video 1 video 2 video 3

15 Sanger sequencing ve +ve Inferred DN sequence Dideoxynucleotide utoradiogram courtesy of Dr. Rahat Zaheer video 1 video 2 video 3

16 typical sequencing/cloning plasmid pu19 is a small, high-copy number E. coli plasmid cloning vector, with multiple cloning sites. he molecule is 2686 bp in length. pu19 encodes the N-terminal fragment of b-galactosidase (lacza), which allows for blue/white colony screening (i.e., a-complementation), as well as a pu origin of replication. he cloned sequence can be amplified by R with M13-47 and RV-M primers or sequenced with either primer. Sino Biological Inc.