dna sequencing
TRANSCRIPT
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EVALUATION SEMINARON
DNA SEQUENCING
PRESENTED BYAMIT SHRESTHA
M-PHARM
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DNADeoxyribonucleic 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. Along with RNA and proteins, DNA is one of the three major
macromolecules essential for all known forms of life.Most DNA molecules are double-stranded helices, consisting
of two long biopolymers of simpler units called nucleotides—each nucleotide is composed of a nucleobase (guanine, adenine, thymine, and cytosine),
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Watson & Crick Model
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DNA SEQUENCING
Determining the order of bases in a section of DNA
four bases—adenine guanine cytosine thymine
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PURPOSE
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms
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DNA SEQUENCING METHODS
Historically there 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.
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MAXAM & GILBERT METHOD
• A. M. Maxam and W.Gilbert-1977
• The sequence of a double-stranded or single-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.
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PRINCIPLE :
Chemical degradation
Reaction in two stages:• Chemical modification of the bases• Modified base is removed from its sugar,
pyperidin cleaves phosphodiester bonds 5’ and 3’ and base is released
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The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced.Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule.The fragments in the four reactions are arranged side by side in geL electrophoresis for size separation.To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred.
PROCEDURE
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SANGER METHOD
• Most common approach used to sequencing DNA.
• Invented by Frederick Sanger - 1977
• Nobel prize - 1980
• Also termed as chain termination or dideoxy method
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SANGER METHOD TERMED AS CHAIN TERMINATION METHOD
This method uses dideoxynucleotide triphosphates(ddNTPs) chain terminators : which have an H on the 3’ carbon of the ribose
sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs).
Therefore in a synthesis reaction, if a
dideoxynucleotide isadded instead of the normal deoxynucleotide, the
synthesisstops at that point because the 3’OH necessary for
theaddition of the next nucleotide is absent.
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DEOXY VERSUS DIDEOXY
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PRINCIPLE :
The sequence of a single-stranded DNA molecule is determined by enzymatic
synthesis of complementary polynucleotide chains.
These chains terminating at specific nucleotide positions.
Separate by gel electrophoresisRead DNA sequence
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REQIREMENTS
DNA sequencing is performed in four separate tubes,
each containing i. Single stranded DNA to be sequenced ii. DNA polymerase iii. Primers iv. The four dNTPs (dATP, dCTP, dTTP and
dGTP) v. Small amount of one of the four ddNTPs (ddATP or ddCTP or ddTTP or ddGTP)
Either the primers or the dNTPs are radiolabeled with 32P
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PROCEDUREIn the Sanger method, the DNA strand to be analyzed is used as a template and DNA polymerase is used, in a PCR reaction, to generate complimentary strands using primers. Four different PCR reaction mixtures are prepared, each containing a certain percentage of dideoxynucleoside triphosphate (ddNTP) analogs to one of the four nucleotides (ATP, CTP, GTP or TTP). Synthesis of the new DNA strand continues until one of these analogs is incorporated, at which time the strand is prematurely truncated. Each PCR reaction will end up containing a mixture of different lengths of DNA strands, all ending with the nucleotide that was dideoxy labeled for that reaction. Gel electrophoresis is then used to separate the strands of the four reactions, in four separate lanes, and determine the sequence of the original template based on what lengths of strands end with what nucleotide.
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COMPARISON
Sanger Method Maxam Gilbert 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
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Applications of DNA Sequencing
• Forensics– Identify individuals– Determine the paternity of a child– Identifies endangered and protected species
• Medicine– Detect genes that are hereditary or cause diseases
• Agriculture– Map the genome of microorganisms
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Future of DNA Sequencing• Projects might focus on researching:
– The links to develop lifestyle– Genomic and cardiovascular disease– Early detections of cancer
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THANK YOU