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DNA SEQUENCING METHOD
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DNA SEQUENCING METHOD

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  • 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.
  • 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.
  • 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.
  • Deciphering “code of life”Detecting mutationsTyping microorganismsIdentifying human halotypesDesignating polymorphisms
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    • 1. DNA SEQUENCING METHODByMuhammad Moazzam AliTrainee Technologist 2013AGA KHAN UNIVERSITY HOSPITAL
    • 2. FutureWho and when it was practiced first?How it is performed?•SummaryWhat is DNA Sequencing method?How does it revolutionize biological sciences?How long will it give benefits?IntroductionHistoryThe MethodAdvantages
    • 3. • Deoxyribonucleic acid (DNA) is a nucleic acid thatfunctions include• Storage of genetic information• Self-duplication & inheritance• Expression of the genetic message• DNA’s major function is to code for proteins. Informationis encoded in the order of the nitrogenous bases.•Deoxyribonucleic AcidThymineGuanineCytosineAdenosine
    • 4. KEY FEATURES OF A DNA• DNA is composed of 2 chains ofnucleotides that form a doublehelix shape.• The two strands are antiparallel.• The backbone of the DNAmolecule is composed ofalternating phosphate groups andsugars.• The complimentary nitrogenousbases form hydrogen bondsbetween the strands.• A is complimentary to T and G iscomplimentary to C.•Watson & Crick Model of DNA
    • 5. •DNA SEQUENCING• Determining the order of bases in asection of DNA• To analyze gene structure and itsrelation to gene expression as well asprotein conformation
    • 6. •PURPOSE• Deciphering “code of life”• Detecting mutations• Typing microorganisms• Identifying human halotypes• Designating polymorphisms
    • 7. •DNA SEQUENCING METHODS•Historically there are two main methods ofDNA sequencing1. Maxam and Gilbert method2. Sanger methodModern sequencing equipment uses theprinciples of the Sanger technique.
    • 8. •A. M. Maxam and W.Gilbert-1977•Chemical Sequencing•Treatment of DNA with certainChemicals  DNA cuts intoFragments  Monitoring ofsequences•MAXAM & GILBERT METHOD
    • 9. •PrincipleA graphical demonstration
    • 10. • Most common approach used forDNA sequencing .• Invented by Frederick Sanger - 1977• Nobel prize - 1980• Also termed as Chain Termination orDideoxy method•SANGER METHOD
    • 11. •SANGER METHOD• The chain termination reaction• Dideoxynucleotide triphosphates (ddNTPs) chainterminators•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
    • 12. •DEOXY VERSUS DIDEOXY
    • 13. •PRINCIPLEssDNAEnzymatic synthesis of complementary polynucleotide chainsTermination at specific nucleotide positionsSeparate by Gel ElectrophoresisRead DNA Sequence
    • 14. •COMPARISONSanger Method Maxam Gilbert MethodEnzymatic ChemicalRequires DNA synthesis Requires DNATermination of chainelongationBreaks DNA at differentnucleotidesAutomation Automation is not availableSingle-stranded DNA Double-stranded or single-stranded DNA
    • 15. •Applications of DNA Sequencing• Forensics: to help identifyindividuals because each individualhas a different genetic sequence• Medicine: can be used to helpdetect the genes which are linked tovarious genetic disorders such asmuscular dystrophy.• Agriculture: The mapping andsequencing of a genome ofmicroorganisms has helped to makethem useful for crops and foodplants.
    • 16. • Advantages• Improved diagnosis of disease• Bio pesticides• Identifying crime suspects• Disadvantages• Whole genome cannot be sequenced at once• Very slow and time consuming
    • 17. •The Human Genome Project• The biggest challenge for the life sciences• 15 years project (NIH, DOE of USA)• Primary goal  Sequence base pairs of human beings that formDNA• Identifying & mapping approx. 20K-25K genes• Significance  Physical & functional•standpoint