Digital Analysis of DNA additional CH 7 information Restriction enzymes fragment the genome at specific sites Each restriction enzyme recognizes a specific sequence of bases anywhere within the genome 1 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 DNA 5 Often palindromic base sequences of each strand are identical 6 Each enzyme cuts at the same place relative to its specific when read 5 to 3 recognition sequence Restriction enzymes RE 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 enzymes Restriction enzymes produce restriction fragments with either blunt or sticky ends Blunt ends cuts are straight through both DNA strands at the line of symmetry Sticky 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 length Average 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 fragments Sites for three restriction enzymes in a 200 kb region of human chromosome 11 Names and location of genes in this region are shown below the restriction sites RsaI 4 base cutter many cuts EcoRI 6 base cutter NotI 8 base cutter few cuts Gel electrophoresis distinguishes DNA fragments according to size Preparing an agarose gel for electrophoresis agarose gel pouring demo from YouTube com Gel 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 charge DNA has negative charge so moves toward the positive Gel electrophoresis distinguishes DNA fragments according to size cont 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 Agarose gel stained with ethidium bromide Migration of DNA fragments Different types of gels separate different sized DNA molecules Polyacrylamide 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 a Do single and double digests with two restriction enzymes b Load each digest into gel along with size markers c Use process of elimination to derive the only possible arrangement that accounts for all the observed fragments Practice restriction mapping problem 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 B 14 8 C 16 6 A x B 8 6 5 3 A x C 11 5 5 1 B x C 8 8 6 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 D 18 5 E 15 7 1 F 20 3 D x E 10 7 5 1 D x F 15 5 3 E x F 15 4 3 1 15 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 PCR consists of repeated cycles of DNA synthesis with three steps in each cycle Two oligonucleotide primers 16 26 nt are needed for PCR reactions Oligonucleotides 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 end of the target region PCR example ADDITIONAL INFORMATION 1 DNA sequence analysis Two methods originally developed Maxam Gilbert method Chemical cleavage of DNA at specific nucleotides Sanger method Enzymatic extension of DNA strands to a defined terminating base Both methods can determine sequence of 750 1000 bp per reaction and have 99 9 accuracy Sanger method is much more amenable to automation Sanger sequencing generates sets of nested fragments separated by size Two steps to the Sanger method 1 From a portion of a template DNA generate a complete series of complementary single stranded subfragments Each subfragment differs in length by a single nucleotide from preceeding and succeeding fragments nested array Each subfragment is defined by its terminal nucleotide 2 Polyacrylamide gel electrophoresis Separates DNA