{[ promptMessage ]}

Bookmark it

{[ promptMessage ]}

L12-13 - L11 start here Berg Tymoczko Stryer Biochemistry...

Info iconThis preview shows pages 1–5. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Biochemistry Sixth Edition Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company Berg • Tymoczko • Stryer Biological processes involve changes in gene expression patterns e.g. metamorphosis •Gene expression can be followed on microarrays. •Pattern changes with changes in biological processes ( e.g. transcription) 5.1) The exploration of genes relies on key tools 1) Restriction enzyme analysis See New England Biolabs <http://www.neb.com> 2) Blotting techniques 3) DNA sequencing At Penn State, <http://www.huck.psu.edu/stf/naf/Sequencing.html> 4) Solid-phase synthesis of nucleic acids At Penn State, <http://www.huck.psu.edu/stf/naf/Synthesis.html> 5) The polymerase chain reaction Interview with Kary Mullis, inventor of PCR and 1993 Nobel Prize in Chemistry: < http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis- interview.html>
Background image of page 1

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

View Full Document Right Arrow Icon
2 1.) Restriction enzymes are DNA scissors Sac II (from S treptomyces ac hromogenes ) in this example 5’CCGC pGG3’ 3’GGp CGCC5’ 2-fold A wide variety of restriction enzymes are commercially available Overhang: 5’ (sticky ends) 5’ (sticky ends) none (flush ends) 3’ (sticky ends) 5’ (sticky ends) •‘Sticky ends’ are aka ‘cohesive ends’ •‘Flush ends’ are aka ‘blunt ends’ Restriction digests of a given DNA can be separated by gels Restriction enzyme: Each enzyme (A, B, C) gives a unique fingerprint. •For shorter DNA (<1 kb), use polyacrylamide gels (PAGE). Denaturing. •For longer DNA (up to 20 kb), use agarose gels. non- denaturing •For very long DNA (1 Mb+), use PFGE (p ulse f ield g el e lectrophoresis)
Background image of page 2
3 2.) Blotting techniques: Southern blotting 20mer radiolabeled ‘*_____’ •Southern blotting was developed by Edwin Southern •Northern blotting is a way to detect an RNA fragment with 32 P-DNA •Western blotting detects proteins with an antibody (see earlier chapters) 3.) Sanger sequencing of DNA •aka ‘dideoxy’ or ‘chain termination’ method •Nobel prize in Chemistry (1980) •Need to run a total of 4 reactions: ddATP, ddCTP, ddGTP, ddTTP molecule 1 molecule 2 (molecule 1 molecule 2) (ddATP is doped in) •missing ‘HO’ •chain has been ‘terminated’ :OH (3’OH of preceding base)
Background image of page 3

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

View Full Document Right Arrow Icon
4 DNA sequencing is automated •DNA sequencing is now done in one pot with fluorescently labeled ddNTP ( ddATP , ddCTP , ddGTP, ddTTP ) •Need to do only 1 (not 4) labeling reactions •Get sequence from 1 (not 4) lanes in a gel •Can sequence 800+ bp at a time (much more efficient than protein sequencing!) 4.) Solid-phase synthesis of DNA (RNA) All nucleophiles are blocked. Each base is protected at two positions: 1.) The 5’-OH 2.) The base itself 3.) For RNA synthesis, must protect 2’OH too! •Trivalent P is highly electrophilic •Must use anhydrous techniques!
Background image of page 4
Image of page 5
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}