dna sequencing – “plus and minus” plus –incubate with t4 dna polymerase and single dntp...
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DNA Sequencing – “Plus and Minus”
• Plus– Incubate with T4 DNA Polymerase and single dNTP– T4 Polymerase degrades 3’ ends in absence of dNTP– Fractionated on PAGE w/ladder
• Minus– Incubate with DNA Polymerase I and 3 dNTPs– Denatured from template– PAGE electrophoresis
• Disadvantage– Neither method completely accurate– Both must be used together
DNA Sequencing – Dideoxy Sequencing
DNA sequencing with chain-terminating inhibitorsF. Sanger, S. Nickelen, and A.R. Coulson (1977)
Sanger et al
• Bacteriophage X174• Restriction enzyme
frag.– A12d and A14
• ddGTP, ddATP, ddTTP and araCTP– Arabinonucleoside
• dATP chase
Sanger et al
• R4 fragment as primer• Digest with restriction
enzyme• ddNTPs used
• Problem– What if second restriction site
close to first one– New bands arise
Sanger et al
• A8 fragment as primer
• Single ribonucleotide incorporated at end of primer
• ddTTP, araCTP, ddCTP, ddGTP, and ddATP
Determining the sequence of both ends of the shotgun library
End-sequencing large-insert clones to find one that spans the region of interest
Shotgun Sequencing (cont.)
• Pilot project for Human Genome
• 2 kbp and 10 kbp mate pairs• BACs were end-sequenced to generate set of mate
pairs• Problem with assembly
– Confusion with repetitive DNA stretches
• Solution – Scaffolds of contigs
Myers et al
• High quality input data needed• Accuracy of pair mate distances• Reliablity of true pairing
The Drosophila Data Sets• WGS – 2 kbp, 10 kbp and BAC• Joint Data Set
– WGS– 2nd and 3rd chromosome– 340 BAC and P1 inserts– X chromosome– 4th chromosome
Myers et al• Screen for rep.
elements– Ribosomal DNA– Histones– Heterochromatin– Retrotransposons
• Overlap: true/repeat induced
Myers et al• Unitigger
– Unique or repetitive seq– Correct subassembly or fragments– Detect repeat boundaries so can remove repetitive overlap
between the unitigs
Myers et al• Scaffolder• Repeat Resolutions
– Rocks - two mate pairs – Stones - single mate pair– Pebbles – find best tiling across gap
• Consensus