DNA Sequencing

? ? DNA extraction PCR Gel electrophoresis Insect identification ACAGATGTCTTGTAATCCGGC CGTTGGTGGCATAGGGAAAG GACATTTAGTGAAAGAAATTG ATGCGATGGGTGGATCGATG GCTTATGCTATCGATCAATCA GGAATTCAATTTAGAGTACTT AATAGTAGCAAAGGAGCTGC TGTTAGAGCAACACGTGCTCA GGCAGATAAAATATTATATCG TCAAGCAATACGTAGTATTCT TGAATATCAAAAATTTTTGTTG GTTATTCA DNA sequencing ACAGATGTC TTGTAATCC GGCCGTTGG TGGCATAGG GAAAGGACA TTTAG Bioinformatics Review of Project

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

2. Amplify and Sequence this region across isolates…. ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG GTFTGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATTTAGAATTCAATTTAGAGTACTTAATAGTAG CAAAGGAGCTGCTGTTAGAGCAACACGTGCTC AGGCAGATAAAATATTATATCGTCAAGCAATAC GT Sequence the PCR product PCR

The elegant idea behind DNA sequencing Technology changes quickly, but for many years we’ve used Sanger’s cool trick. Fred Sanger 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

DNA Polymerase Action

Mechanism of DNA polymerization

Dideoxy Nucleotide

Dideoxies block elongation H ddGTP X

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.

Dideoxy (Sanger) Method 4 Steps: 1.Denaturation 2.Primer attachment and extension of bases (starting point) 3.Termination 4.Gel electrophoresis

Overview: Dideoxy (Sanger) Method Gel electrophoresis 5 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

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

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

DNA Sequencing Reactions

Gel Electrophoretic Fractionation of Products

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.

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.

P.S: before coming the class, please watch the link WEtc