Bio118 Genomics NOOR-1

Bio118 Genomics NOOR-1 - Genomics Restriction enzymes...

Info iconThis preview shows pages 1–14. Sign up to view the full content.

View Full Document Right Arrow Icon
Restriction enzymes Cloning Polymerase Chain Reaction DNA sequencing Whole genome shotgun sequencing Polymorphic molecular markers Reverse transcription Transcription profiling Genomics Goal: Understand how technologies work Understand what is possible Improved understanding of biological mechanisms and processes
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Objectives… Have an organism from which we’ve isolated DNA Desire to Focus on particular (or random) segments Amplify those segments for manipulation or to get sequences Put it all together Identify variation among individuals Look at what is transcribed and how much
Background image of page 2
Objectives… Have an organism from which we’ve isolated DNA Desire to Focus on particular (or random) segments Amplify those segments for manipulation or to get sequences Put it all together Identify variation among individuals Look at what is transcribed and how much
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Restriction Enzymes or Restriction Endonucleases Cuts double-stranded DNA at a specific recognition site http://en.wikipedia.org EcoRI recognition site Enables recombinant DNA technology (cloning) Cut DNA into manageable size Palindrome – same nt sequence in antiparallel orientation
Background image of page 4
Cloning Plasmid: circular DNA molecule that can replicate in bacteria; Often “man- made” with known cut-sites Ligation Restriction digestion
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Objectives… Have an organism from which we’ve isolated DNA Desire to Focus on particular (or random) segments Amplify those segments for manipulation or to get sequences Put it all together Identify variation among individuals Look at what is transcribed and how much
Background image of page 6
PCR: Polymerase Chain Reaction Cyclical process with three stages Denaturation 94 o C Annealing 50-55 o C Extension 72 o C Iterated repeatedly, results in exponential amplification of a specific, targeted DNA sequence!
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Polymerase Chain Reaction - PCR 5’-A CGTA GACGTTAGCTAACGCGTT ACGA T-3’ 3’-TGCATCTGCAATCGATTGCGCAATGCTA-5’ 5’-ACGTAGACGTTAGCTAACGCGTTACGAT-3’ 3’-TGCT-5’ 5’-CGTA-3’ Forward primer: 5’-CGTA-3’ Reverse primer: 5’-TCGT-3’ Real PCR primers are ~20 nt (underlined sequences are “previously known”)
Background image of page 8
Polymerase Chain Reaction - PCR Specificity depends on primers
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Polymerase Chain Reaction - PCR
Background image of page 10
Technology determines what is feasible: Sequencing involves a chemical reaction (1) First need to have made enough DNA to detect (e.g., cloning, PCR) (2) Synthesize new DNA strand in vitro (requires one primer to begin) (3) Method to detect each base that is added DNA sequencing
Background image of page 11

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Sanger sequencing method = in vitro DNA replication but with “terminator” dideoxy NTPs (ddNTPs) Randomly stop reaction because lack the 3’-OH that DNA polymerase requires for chain elongation Sanger sequencing can only determine about 900 nucleotides at a time (per “read”)
Background image of page 12
DNA pol I copies template starting at primer random incorporation of ddNTP stops chain
Background image of page 13

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 14
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 55

Bio118 Genomics NOOR-1 - Genomics Restriction enzymes...

This preview shows document pages 1 - 14. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online