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A  restriction map  is a map of known restriction sites within a sequence of DNA

RESTRICTION MAPPING . the process of obtaining structural information on a piece of DNA by the use of restriction enzymes. A  restriction map  is a map of known restriction sites within a sequence of DNA. Restriction mapping steps Breaking DNA into pieces

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A  restriction map  is a map of known restriction sites within a sequence of DNA

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  1. RESTRICTION MAPPING • the process of obtaining structural information on a piece of DNA by the use of restriction enzymes. A restriction map is a map of known restriction sites within a sequence of DNA Restriction mapping steps Breaking DNA into pieces identifying the locations of the breakpoints.

  2. Restriction Enzymes • endonucleases that recognize specific 4 to 8 base regions of DNA. restriction sites. • evolved as a bacterial defense against DNA bacteriophage • Recognizes Palindromicseq, • each strand of the DNA can self-anneal and the DNA forms a small cruciform structure • Hundreds of restriction enzymes that have been isolated and each one recognizes its own specific nucleotide sequence.

  3. Sites for each restriction enzyme are distributed randomly throughout a particular DNA stretch. Digestion of DNA by restriction enzymes is very reproducible; every time a specific piece of DNA is cut by a specific enzyme, the same pattern of digestion will occur.

  4. Uses of Restriction Mapping for many techniques used to manipulate DNA. One application is to cut a large piece of DNA into smaller fragments to allow it to be sequenced. Genes and cDNAs can be thousands of kilobases long (megabases - Mb); however, they can only be sequenced 400 bases at a time. DNA must be chopped up into smaller pieces and sub cloned to perform the sequencing.

  5. an easy way to compare DNA fragments without having any information of their nucleotide sequence.

  6. The sum of the individual fragments =size of the original fragment • If not there are two likely problems. • In one case, some of the smaller fragments may have run off the end of the gel. • the gel was not dense enough and therefore was unable to resolve fragments close in size. • This leads to a lack of separation of fragments which were close in size.

  7. Course Contents • DNA and RNA isolation • Quantification of DNA and RNA • Primer designing • PCR • Electrophoresis • Sequencing • Karyotyping • Restriction Mapping • Flow cytometry • Hybridization • Western blotting • Southern blotting • Northern blotting • FISH

  8. Tissue culturing • Slide Preparation and Cell Stains • Agar plate preparation and streaking for the purpose of individual colony isolation • Bacterial Growth on selective agar • Quantification: Colony Forming Units (CFU) • Dilution Plating • Identification and characteristics of colonies • Transfection • Transduction • Transformation • Cloning • Microarrays • Chromatography • Immunochemistry • ELISA • Bioinformatics and techniques • Ethical issues

  9. Lab Work: • DNA extraction • Quantification • PCR • Electrophoresis • ELISA • Agar plate preparation • Streaking • Transfection • Cloning

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