Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition

Techniques for DNA Study 1.Cleavage and mapping using restriction enzymes. 2.Blotting techniques. Southern: DNA Northern: RNA Western: Protein 3.DNA sequencing. Gilbert-Maxam: degradative method Sanger: synthetic method 4.Solid-phase DNA synthesis. 5.Polymerase chain reaction

Sac I Cleavage

Restriction Enzymes Recognition and Cleavage sites Blunt ends vs Sticky ends

Southern Blotting Detection of a specific DNA Fragment

Northern & Western Blotting Northern blotting: Detection of a specific RNA Electrophoresis of a mixture of RNAs on agarose Blot onto nitrocellulose Hybridize with a specific DNA or RNA sequence Detect by autoradiography. Western blotting: Detection of a specific protein Electrophoresis of a protein mixture (SDS-PAGE) Blot onto nitrocellulose Hybridize with an antibody specific to the protein Detect by autoradiography.

Restriction Enzymes These are endonucleases whose purpose is to restrict expression of foreign DNA, i.e. to protect a bacteria from a bacteriophage. Type II enzymes are commonly used. They need no ATP and cleave 4-8 bp within recognition site. No cleavage occurs at a restriction site if it is methylated. The host uses a restriction methylase to alter one DNA strand, this protects the host DNA. (Genus = 1st letter; Species = next two; Strain; Number known = Roman numeral) E.g. EcoRIEscherichia coli R

Mapping with Type II Enzymes Electrophoresis of restriction fragments from a 4.0 kb pair segment of DNA with P 32 on 5 ’ end P 32

Mapping with Type II Enzymes Individual and combined maps Select Overlap

DNA Sequencing Sanger developed the synthetic “dideoxy” method:This method uses 2',3'-dideoxy- ribonucleotidetriphosphates to stop synthesis at random points such that all possible fragments are generated. Gilbert-Maxam is a degradation method: It was developed before the Sanger method and uses selective cleavage of DNA to produce all possible fragments.

Sequencing by Sanger’s dideoxy method

Sanger’s dideoxy method Start with a fragment of ssDNA to be sequenced (template) and primer piece to initiate synthesis. Add the 4 dNTP's plus a trace of radiolabel. Then divide the solution into 4 samples. Add a trace of: ddATP to 1, ddGTP to 2, ddCTP to 3, ddTTP to 4. This allows random incorporation of the ddNTP where bases pair. Treat each of these four samples with the Klenow fragment of DNA Polymerase I (Pol I).

Sanger’s dideoxy method Pol I catalyzes polymerization of the nucleotides making a DNA complement of the template. Polymerization halts on incorporation of ddNTP. The samples are subjected to side-by-side electrophoresis (parallel lanes). Bands are observed by autoradiography and yield the sequence of template’s complement. Alternatively a fluorescent label can be used.

Electrophoresis

Fluorescence detection This separation is by capillary electrophoresis. As each band exits the capillary column detection is by fluorescence spectroscopy.

Solid-phase DNA Synthesis

Polymerase Chain Reaction Method of amplifying (making many copies of) a DNA segment. 1.Mix template, primers, dNTPs and DNA polymerase 2.Heat to 95 o C to separate strands (denature) 3.Cool to 54 o C to anneal primers 4.Warm to 74 o C for optimum polymerase activity (synthesis occurs 5 ’ to 3 ’ ) 5.Repeat steps 2-4.

5’5’ 3’3’ 5’5’ 3’3’

End of Chapter 5 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition