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DNA Lab: Restriction Mapping Cell and Molecular Biology 1 Lab #3 Autumn 2005 DNA Mapping Lab DNA Lab: Restriction mapping Readings in Alberts 2nd Ed (Ch 10: 328-330; 341-345); Becker 6th Ed (Ch18: 524-527; Ch 20: 633-640); 5th Ed (Ch16: 492-496; Ch 18: 608-610). Useful Web sites Primer on mapping DNA: http://www.ncbi.nlm.nih.gov/About/primer/mapping.html Tutorial on restriction mapping: http://www.carolina.com/biotech/plasmid_problems/plasmid_guide.asp Within the 3 billion or so nucleotides that make up the human genome, identifying one particular gene that is composed of only several thousand nucleotides is a challenging task. This task is simplified somewhat by creating maps of the DNA. Just like a road map, a DNA map uses DNA dependent landmarks to help determine where a gene resides in the genome. DNA can be mapped to varying degrees of resolution. Genetic maps are usually lower resolution maps, made by measuring the recombination frequency between different heritable traits. Genetic maps are used to associate a region of a chromosome with a particular trait; they also can be used to determine gene order along a chromosome. Physical (distance) maps are higher resolution maps based on DNA sequence, and are measured in numbers of base pairs (often in kilobases or megabases). The ultimate physical map of a piece of DNA is its complete sequence. However, if DNA sequence information is not available, a lower resolution map can still be generated using enzymes that cut DNA at specific sequences. These enzymes are called restriction enzymes. Restriction mapping is used to determine the distance between the sites where restriction enzymes cut and to order the different sites relative to each other. The cut sites serve as landmarks when keeping track of several pieces of DNA. Genetic and physical maps can be correlated to identify the DNA sequences encoding a particular trait. Beyond mapping, restriction enzymes also are useful in recombinant DNA technology. Two different DNA pieces cut by the same restriction enzyme can be readily joined back together in a test tube. Moreover, because the genetic code is universal, it is possible to join DNA from one organism to the DNA of another organism and produce a functional gene. Recall from the Fluorescence Microscopy lab where GFP was targeted to the mitochondria of tobacco cells. DNAs from four different organisms were incorporated into the tobacco cells. A short segment of the yeast coxIV gene was joined to the jellyfish GFP gene in a test tube; the fusion gene was then cloned into a transformation vector that contained sequences from bacteria and from a plant virus. In this lab, you will be mapping some chloroplast genes isolated from the single-celled alga, Chlamydomonas. The chloroplast genome was broken up into smaller pieces called clones; your goal is to put together a restriction map for one of these clones. Based on the restriction enzyme pattern for each clone, it is possible to reconstruct the entire map of the chloroplast DNA. Please read through the background material so you will understand some common terms in molecular biology. Terms to understand: Agarose gel electrophoresis Ethidium Bromide Multiple cloning site (MCS) Physical map Plasmid Restriction enzyme Restriction map Vector Cell and Molecular Biology 2 Lab #3 Autumn 2005 DNA Mapping Lab Background Restriction Endonucleases Restriction endonucleases are enzymes that cut DNA at specific sequences. For example, the enzyme EcoRI cuts DNA at the sequence GAATTC. The enzymes hydrolyze the phosphate backbone creating a nick in the DNA strand. Bacteria produce restriction enzymes as a defense against invading viral DNA. In E. coli, foreign DNA with the sequence GAATTC would be cut and inactivated. To protect its own DNA from the restriction enzyme, bacteria also produce DNA modifying enzymes. For each restriction endonuclease there is a corresponding modifying enzyme that blocks restriction activity on the host’s own DNA, generally by methylating the DNA at the recognition sequence. The protruding methyl group presumably prevents catalysis by interfering with the close molecular interaction between the restriction enzyme and its recognition site. For example, EcoRI methylase adds a methyl group to the second adenine residue within the EcoRI recognition site (GAATTC). The discovery of restriction enzymes is a wonderful lesson on the unexpected outcomes of basic research. The enzymes were discovered by scientists studying the infection of bacteria by viruses. As the scientists began to understand how bacteria protected themselves against viruses using these enzymes, the idea of using restriction enzymes for manipulating other DNAs was developed. Research on restriction enzymes led to a Nobel prize for Arber, Nathans, and Smith in 1978. Several hundred restriction enzymes have been isolated from prokaryotic organisms, and many are commercially available. Restriction enzyme names follow a standard nomenclature system: • The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated. • The second and third letters are the initial letters of the organism’s species name. • A fourth letter, if any, indicates a particular strain of organism. • Roman numerals indicate the sequence in which different endonucleases were isolated from a particular organism and strain. EcoRI HindIII E = genus Escherichia H = genus Hemophilus co = species coli in = species influenzae R = strain RY13 d = strain d I = first endonuclease isolated III = third endonuclease isolated Each restriction endonuclease scans along a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides. Most restriction enzymes recognize a four- or six-base pair (bp) sequence. At or near the recognition site, the enzyme catalyzes a hydrolysis reaction that breaks the phosphodiester linkage on each strand of the DNA helix. Two DNA fragments are produced, each with a phosphate at the 5' end and a hydroxyl at the 3’ end. Restriction enzymes cut both strands of the double helix. In some cases, the enzyme cuts at the midpoint of the recognition sequence. These enzymes leave DNA fragments with blunt-ends. Example of blunt end digestion by EcoRV (recognition sequence GATATC) 5'- N-N-G-A-T-A-T-C-N-N-3' 5'-N-N-G-A-T + A-T-C-N-N-3' 3'- N-N-C-T-A-T-A-G-N-N-5' 3'-N-N-C-T-A T-A-G-N-N-5' Other endonucleases cleave each strand off-center in the recognition site, at positions two-to-four nucleotides apart, creating fragments with exposed single-stranded ends. This leaves single-stranded “overhangs” on either the 5’ or 3’ ends of the DNA fragments. Single-stranded overhangs, also called “sticky” ends, are useful in making recombinant DNA molecules because they can hydrogen bond to each other efficiently. For example, EcoRI recognizes the six-base sequence GAATTC and it cuts leaving a 5’ overhang of four nucleotides (AATT). Example of staggered (sticky) end digestion by EcoRI (recognition sequence GAATTC) 5'- N-N-G-A-A-T-T-C-N-N-3' 5'-N-N-G + A-A-T-T-C-N-N-3' Cell and Molecular Biology 3 Lab #3 Autumn 2005 DNA Mapping Lab 3'- N-N-C-T-T-A-A-G-N-N-5' 3'-N-N-C-T-T-A-A G-N-N-5' Regardless of the source of DNA - whether from prokaryote or eukaryote - EcoRI will always cut at the GAATTC recognition site and it will always produce fragments with 5'-AATT overhangs. When the enzyme is removed, however, the overhangs can hydrogen bond (anneal) to each because of complementary base-pairing. Any EcoRI fragment can anneal to any other EcoRI fragment. After annealing, the phosphate backbone of the two DNA strands can be repaired by the enzyme DNA ligase. These steps are the basis of recombinant DNA technology. For example, if an EcoRI fragment of a yeast gene is annealed and ligated to a jellyfish GFP gene EcoRI fragment, the resulting chimeric DNA would be a novel part yeast-part jellyfish gene. Recombination of two EcoRI fragments note the ligated backbone 5'-N-G 5'-A-A-T-T-C-X-3' 5'-N-G A-A-T-T-C-X-3' 5'-N-G-A-A-T-T-C-X-3' 3'-N-C-T-T-A-A G-X-5' 3'-N-C-T-T-A-A G-X-5' 3'-N-C-T-T-A-A-G-X-5' yeast EcoRI GFP EcoRI Annealed EcoRI Ligated EcoRI sites fragment fragment sites Agarose Gel Electrophoresis Digestion of a large piece of DNA with a restriction enzyme will generate smaller DNA fragments, whose sizes can be determined - - - by gel electrophoresis. Electrophoresis literally means, "to carry O O O by electricity". In gel electrophoresis, molecules are separated | | | based on their charge and their size. DNA, an organic acid, is a O-P-O-R-O-P-O-R-O-P-O highly negatively charged molecule due to the phosphate || || || backbone. In solution, hydrogen ions are liberated from the O O O phosphate groups, leaving negatively-charged oxygen ions radiating along the outside of the DNA molecule. When placed within an electrical field, the negatively charged DNA molecules are attracted toward the positive pole and repelled from the negative pole. DNA migration takes place through a gel matrix that acts as a molecular sieve to sort restriction fragments by size. The two most common matrix materials are agarose, a highly purified form of agar, and polyacrylamide, a synthetic polymer. As DNA fragments move though the pores in the matrix toward the positive pole of the electrical field, the gel matrix (“the sieve”) impedes the movement of larger fragments. Fragment mobility is inversely proportional to molecular weight (number of base pairs).
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