Dna Sequencing-4th Level
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DNA Sequencing
Overview of Sequencing • Most powerful methods ever devised for DNA analysis • It is used to: – – – –
Determine the precise order of base sequence Determine all restriction sites within a gene Predict protein product Provide information about potential sites of introns in genome – Gives information about mechanism of expression of a gene
Two Methods
Chemical Cleavage Maxam-Gilbert
Enzymatic Cleavage Sanger
Chemical Cleavage Maxam-Gilbert 1970, based on chemical cleavage of restriction fragments. The fragments are end-labeled with radioactive nuclei. The resulting sub fragments are separated by gel electrophoresis and labeled fragments are detected by autoradiography.
Walter Gilbert
Fredrick sanger
Mary-Clair King- BRCA 1
Enzymatic Cleavage Sanger Based on random termination of a DNA chain during enzymatic synthesis. The technique is possible because the dideoxy analogue of each of the four normal nucleotides can be incorporated into a growing chain by DNA polymerase.
Cell-Based Cloning (1) Case: A thirteen year old girl is admitted with dehydration, vomiting & weight loss. Her blood glucose level is 344mg/dL (19.1mmol/L) and she has ketonuria. A diagnosis of type 1 diabetes mellitus. She is started on recombinant human insulin, is rehydrated, and makes a prompt recovery.
Cell-Based Cloning (2) •Previously insulin therapy involved the use of animal insulins, most commonly pork or beef, which were chemically similar, but not identical, to human insulin. •Animal insulins often led to the development of anitbodies, which reduced the efficacy of the insulin and could lead to treatment failures. •Following the cloning of the human insulin gene, large scale production of pure human insulin was performed by inserting the cloned gene into an invitro amplification system. •Thus, large amounts of insulin gene copies may be produced, which are then expressed in either bacteria or yeast and the resulting purified insulin is then available for use in diabetic patients.
Automated Gene Sequencing • The human genome project’s aim is to obtain the DNA sequence of the entire human genome. • By product of this project was the development of technology such as automation of the sequencing process. • Automated DNA sequencing uses fluorescent rather than radio-labeled products. • Using four different fluorescent tags, one for each sequencing reaction, resulted in – – – –
All four reactions can run on the same lane Scanner device can then read the gel Enables four times as many reactions to be performed for gel Autoradiography time is abolished.
End Of Show
Overview of Sequencing • Most powerful methods ever devised for DNA analysis • It is used to: – – – –
Determine the precise order of base sequence Determine all restriction sites within a gene Predict protein product Provide information about potential sites of introns in genome – Gives information about mechanism of expression of a gene
Two Methods
Chemical Cleavage Maxam-Gilbert
Enzymatic Cleavage Sanger
Chemical Cleavage Maxam-Gilbert 1970, based on chemical cleavage of restriction fragments. The fragments are end-labeled with radioactive nuclei. The resulting sub fragments are separated by gel electrophoresis and labeled fragments are detected by autoradiography.
Walter Gilbert
Fredrick sanger
Mary-Clair King- BRCA 1
Enzymatic Cleavage Sanger Based on random termination of a DNA chain during enzymatic synthesis. The technique is possible because the dideoxy analogue of each of the four normal nucleotides can be incorporated into a growing chain by DNA polymerase.
Cell-Based Cloning (1) Case: A thirteen year old girl is admitted with dehydration, vomiting & weight loss. Her blood glucose level is 344mg/dL (19.1mmol/L) and she has ketonuria. A diagnosis of type 1 diabetes mellitus. She is started on recombinant human insulin, is rehydrated, and makes a prompt recovery.
Cell-Based Cloning (2) •Previously insulin therapy involved the use of animal insulins, most commonly pork or beef, which were chemically similar, but not identical, to human insulin. •Animal insulins often led to the development of anitbodies, which reduced the efficacy of the insulin and could lead to treatment failures. •Following the cloning of the human insulin gene, large scale production of pure human insulin was performed by inserting the cloned gene into an invitro amplification system. •Thus, large amounts of insulin gene copies may be produced, which are then expressed in either bacteria or yeast and the resulting purified insulin is then available for use in diabetic patients.
Automated Gene Sequencing • The human genome project’s aim is to obtain the DNA sequence of the entire human genome. • By product of this project was the development of technology such as automation of the sequencing process. • Automated DNA sequencing uses fluorescent rather than radio-labeled products. • Using four different fluorescent tags, one for each sequencing reaction, resulted in – – – –
All four reactions can run on the same lane Scanner device can then read the gel Enables four times as many reactions to be performed for gel Autoradiography time is abolished.
End Of Show
