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DNA SEQUENCING METHOD
DNA sequencing determines the order of nucleotide bases in a DNA molecule. The first methods were developed in the 1970s by Maxam and Gilbert (chemical method) and Sanger (chain termination/dideoxy method). Sanger's method is now most commonly used and involves DNA synthesis with chain termination by dideoxynucleotides to generate fragments of different lengths that can be separated and read to determine the DNA sequence. DNA sequencing has revolutionized biological sciences by enabling diagnosis of genetic diseases, identification of disease-causing mutations, and mapping of genomes. It provides benefits for medicine, forensics, and agriculture.
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DNA SEQUENCING METHOD
- 2. DNA SEQUENCING METHOD By MuhammadMoazzam Ali Trainee Technologist 2013 AGA KHAN UNIVERSITY HOSPITAL
- 3. Future Who and whenit was practiced first? How it is performed? •Summary What is DNA Sequencing method? How does it revolutionize biological sciences? How long will it give benefits? Introduction History The Method Advantages
- 4. • Deoxyribonucleic acid(DNA) is a nucleic acid that functions include • Storage of genetic information • Self-duplication & inheritance • Expression of the genetic message • DNA’s major function is to code for proteins. Information is encoded in the order of the nitrogenous bases. •Deoxyribonucleic Acid ThymineGuanineCytosineAdenosine
- 5. KEY FEATURES OFA DNA • DNA is composed of 2 chains of nucleotides that form a double helix shape. • The two strands are antiparallel. • The backbone of the DNA molecule is composed of alternating phosphate groups and sugars. • The complimentary nitrogenous bases form hydrogen bonds between the strands. • A is complimentary to T and G is complimentary to C. •Watson & Crick Model of DNA
- 6. •DNA SEQUENCING • Determiningthe order of bases in a section of DNA • To analyze gene structure and its relation to gene expression as well as protein conformation
- 7. •PURPOSE • Deciphering “codeof life” • Detecting mutations • Typing microorganisms • Identifying human halotypes • Designating polymorphisms
- 8. •DNA SEQUENCING METHODS •Historicallythere are two main methods of DNA sequencing 1. Maxam and Gilbert method 2. Sanger method Modern sequencing equipment uses the principles of the Sanger technique.
- 9. •A. M. Maxamand W.Gilbert-1977 •Chemical Sequencing •Treatment of DNA with certain Chemicals DNA cuts into Fragments Monitoring of sequences •MAXAM & GILBERT METHOD
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- 11. • Most commonapproach used for DNA sequencing . • Invented by Frederick Sanger - 1977 • Nobel prize - 1980 • Also termed as Chain Termination or Dideoxy method •SANGER METHOD
- 12. •SANGER METHOD • Thechain termination reaction • Dideoxynucleotide triphosphates (ddNTPs) chain terminators •havig an H on the 3’C of the ribose sugar (normally OH found in dNTPs) • ssDNA addition of dNTPs elongation • ssDNA addition of ddNTPs elongation stops
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- 14. •PRINCIPLE ssDNA Enzymatic synthesis ofcomplementary polynucleotide chains Termination at specific nucleotide positions Separate by Gel Electrophoresis Read DNA Sequence
- 16. •COMPARISON Sanger Method MaxamGilbert Method Enzymatic Chemical Requires DNA synthesis Requires DNA Termination of chain elongation Breaks DNA at different nucleotides Automation Automation is not available Single-stranded DNA Double-stranded or single- stranded DNA
- 17. •Applications of DNASequencing • Forensics: to help identify individuals because each individual has a different genetic sequence • Medicine: can be used to help detect the genes which are linked to various genetic disorders such as muscular dystrophy. • Agriculture: The mapping and sequencing of a genome of microorganisms has helped to make them useful for crops and food plants.
- 18. • Advantages • Improveddiagnosis of disease • Bio pesticides • Identifying crime suspects • Disadvantages • Whole genome cannot be sequenced at once • Very slow and time consuming
- 19. •The Human GenomeProject • The biggest challenge for the life sciences • 15 years project (NIH, DOE of USA) • Primary goal Sequence base pairs of human beings that form DNA • Identifying & mapping approx. 20K-25K genes • Significance Physical & functional •standpoint
Editor's Notes
- #5 DNA Deoxyribonucleic acid (DNA) is a nucleic acid that functions include Storage of genetic information Self-duplication & inheritance. Expression of the genetic message . DNA’s major function is to code for proteins. Information is encoded in the order of the nitrogenous bases.
- #6 Watson & Crick Model DNA is composed of 2 chains of nucleotides that form a double helix shape. The two strands are antiparallel. The backbone of the DNA molecule is composed of alternating phosphate groups and sugars. The complimentary nitrogenous bases form hydrogen bonds between the strands. A is complimentary to T and G is complimentary to C.
- #7 DNA SEQUENCING Determining the order of bases in a section of DNA. To analyze gene structure and its relation to gene expression as well as protein conformation.
- #8 Deciphering “code of life”Detecting mutationsTyping microorganismsIdentifying human halotypesDesignating polymorphisms