Slide 1Restriction enzymes fragment the genome at specific sitesRestriction enzymesTen commonly used restriction enzymesSlide 5Slide 6Slide 7Slide 8Slide 9Slide 10Agarose gel stained with ethidium bromideDifferent types of gels separate different-sized DNA moleculesSlide 13Deducing a restriction mapPractice restriction mapping problemSlide 16Slide 17Slide 18DNA sequence analysisSlide 20Sanger sequencingSlide 22Slide 23Slide 24Automated DNA sequencingSlide 26Accumulation of genome sequence dataSlide 28Digital Analysis of DNA(additional CH 7 information)Restriction enzymes fragment the genome at specific sitesEach restriction enzyme recognizes a specific sequence of bases anywhere within the genome1. Recognize PALINDROMIC sequences (i.e. Madam, I’m Adam)2. Cuts sugar-phosphate backbones of both strands.3. Restriction fragments are generated by digestion ofDNA with restriction enzymes.4. Hundreds of restriction enzymes are now available.Recognition sites for restriction enzymes are usually 4 – 8 bp of double-strand DNA5. Often palindromic – base sequences of each strand are identical when read 5’to 3’.6. Each enzyme cuts at the same place relative to its specific recognition sequence.Restriction enzymesRE are found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the RE selectively cut up foreign DNA; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity.Ten commonly used restriction enzymesRestriction enzymes produce restriction fragments with either blunt or sticky endsBlunt ends – cuts are straight through both DNA strands at the line of symmetrySticky ends – cuts are displaced equally on either side of line of symmetry.•Ends have either 5‘ overhangs or 3’ overhangs.•Ideal for inserting DNA (cloning)Different restriction enzymes produce fragments of different lengthAverage fragment length is 4n, where n is the number of bases in the recognition site•4-base recognition site occurs every 44 bp, average restriction fragment size is 256 bp3 billion bp genome/256 = 12 million fragments•6-base recognition site occurs every 46 bp, average restriction fragment size is 4100 bp (4.1 kb)3 billion bp genome/4100 = 700,000 fragments•8-base recognition site occurs every 48 bp, average restriction fragment size is 65,500 bp (65.5 kb)3 billion bp genome/65,500 = 46,000 fragmentsSites for three restriction enzymes in a 200 kb region of human chromosome 11Names and location of genes in this region are shown below the restriction sites.RsaI = 4 base cutter (many cuts)EcoRI = 6 base cutterNotI = 8 base cutter (few cuts)Gel electrophoresis distinguishes DNA fragments according to sizePreparing an agarose gel for electrophoresisagarose gel pouring demo from YouTube.comGel electrophoresis distinguishes DNA fragments according to size (cont.)Load DNA samples into wells in gel, place gel in buffered aqueous solution, and apply electric current.•Electrophoresis (movement of charged particles in an electric field)•DNA has negative charge, so moves toward the positive charge.• With linear DNA fragments, migration distance through gel depends on size.• Circular DNA does not migrate the same as linear fragments.• After electrophoresis, visualize DNA fragments by staining gel wit fluorescent dye, and photograph gel under UV light.• Determine size of unknown fragments by comparison to migration of DNA makers of known size.Gel electrophoresis distinguishes DNA fragments according to size (cont.)Agarose gel stained with ethidium bromideMigration of DNA fragmentsDifferent types of gels separate different-sized DNA moleculesPolyacrylamide gels (left) separate small fragments (fine resolution, down to single bp differences)Agarose gels (right) separate larger fragments (> 50 bp fragments)Restriction maps provide sequence-specific landmarks in the DNA terrain• Restriction maps show the relative orders and distances between multiple restriction sites.• Construction of restriction map:1. Digest DNA sample with different restriction enzymes, single digests vs. double digests.2. Run gel and determine fragment sizes for each digest.3. Deduce restriction arrangement of sites by process of elimination.Deducing a restriction map(b) Load each digest into gel along with size markers.(a) Do single and double digests with two restriction enzymes.(c) Use process of elimination to derive the only possible arrangement that accounts for all the observed fragments.Practice restriction mapping problem15“A”, “B” and “C” represent different enzymes. So, using enzyme “B”, you would generate 2 fragments; one 14 and one 8 in length. Likewise, using enzyme “C”, you would generate 2 fragments; one 16 and one 6 in length. You need to figure out WHERE the restriction sites are located in a linear piece of DNA based on the fragments generated.“A”, “B” and “C” represent different enzymes. So, using enzyme “B”, you would generate 2 fragments; one 14 and one 8 in length. Likewise, using enzyme “C”, you would generate 2 fragments; one 16 and one 6 in length. You need to figure out WHERE the restriction sites are located in a linear piece of DNA based on the fragments generated.In each problem set below (defined by the red boxes), three restriction enzymes (A,B,C and D,E,F) are used to cut a piece of DNA, singly and in pairwise combinations. Sizes of fragments are listed in order of size, not in linear order. Determine the correct order of restriction sites, and draw the map, with the intervals between sites labeled.A) 11, 6, 5 D) 18, 5 B) 14,8 E) 15, 7, 1 C) 16,6 F) 20, 3 A x B) 8, 6, 5, 3 D x E) 10, 7, 5, 1A x C) 11, 5, 5,1 D x F) 15, 5, 3 B x C) 8, 8, 6 E x F) 15, 4, 3, 1PCR consists of repeated cycles of DNA synthesis, with three steps in each cycleTwo oligonucleotide primers (16 – 26 nt) are needed for PCR reactionsOligonucleotides are designed from previously known DNA sequence and serve as primers for DNA synthesis.•Target sequence located between primer sequences are exponentially amplified by 25-30 cycles of DNA synthesis.Region between the two primers will be synthesized.•One primer is complementary to one strand of DNA at the end of the target region.•The other primer is complementary to the other strand of Dna at the other