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DNA Sequencing

DNA Sequencing. Bioinformatics. ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAG. Review of Project. PCR. DNA extraction. Insect identification. ?. DNA sequencing. Gel electrophoresis.

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DNA Sequencing

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  1. DNA Sequencing

  2. Bioinformatics ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAG Review of Project PCR DNA extraction Insect identification ? DNA sequencing Gel electrophoresis ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCA

  3. How is this organism related to other species? K ? C A H F I J D B E G

  4. ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT PCR Sequence the PCR product ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGGTFTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATTTAGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT 2. Amplify andSequence this region across isolates….

  5. Fred Sanger The elegant idea behind DNA sequencing Technology changes quickly, but for many years we’ve used Sanger’s cool trick. In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators or dideoxynucleotides. This method is still in use today. What is the basis of Sanger’s method? Shared with Walter Gilbert and Paul Berg

  6. DNA Polymerase Action

  7. Mechanism of DNA polymerization

  8. Dideoxy Nucleotide

  9. ddGTP X H Dideoxies block elongation

  10. Sanger Sequencing • Uses DNA polymerase to synthesize a second DNA strand that is labeled. • DNA polymerase always adds new bases to the 3’ end of a primer that is base-paired to the template DNA. • chain terminator nucleotides: dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3' carbon of the deoxyribose. • When DNA polymerase inserts one of these ddNTPs into the growing DNA chain, the chain terminates, as nothing can be added to its 3' end.

  11. Dideoxy (Sanger) Method • 4 Steps: • Denaturation • Primer attachment and extension of bases (starting point) • Termination • Gel electrophoresis

  12. Overview: Dideoxy (Sanger) Method reactions contain the 4 normal dNTPs, but each reaction also contains one of the ddNTPs. In each reaction, DNA polymerase starts creating the second strand beginning at the primer. When DNA polymerase reaches a base and the chain will either: - terminate if a ddNTP is added, or: continue if the corresponding dNTP is added. - which one happens is random, based on ratio of dNTP to ddNTP in the tube 2 3 1 4 Gel electrophoresis 5

  13. Dideoxy (Sanger) Method • ddNTP- 2’,3’-dideoxynucleotide • No 3’ hydroxyl • Terminates chain when incorporated • Add enough so each ddNTP is randomly and completely incorporated at each base

  14. Dideoxy Method • Run four separate reactions each with different ddNTPs • Run on a gel in four separate lanes • Read the gel from the bottom up

  15. DNA Sequencing Reactions

  16. Gel Electrophoretic Fractionationof Products

  17. Automated Version of the Dideoxy Method Automated sequencers use 4 different fluorescent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the same lane of the gel.

  18. Quality of sequence data may vary, depending on: • Purity and concentration of template DNA • Presence of extra PCR bands (artifacts) • Quality of dye-terminators, electrophoresis matrix, and other reagents Ideally, look at chromatograms and convince yourself that base calls are robust.

  19. P.S: before coming the class, please watch the link http://www.youtube.com/watch?v=bEFLBf5WEtc

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