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DNA Technology and Genomics Chapter 20. A. P. Biology Mr. Knowles Liberty Senior High School. Restriction Endonucleases. Enzymes that recognize specific DNA sequences in dsDNA (usually 4-6 bp sequence of nucleotides) and digest the DNA at this site.
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DNA Technology and GenomicsChapter 20 A. P. Biology Mr. Knowles Liberty Senior High School
Restriction Endonucleases • Enzymes that recognize specific DNAsequences in dsDNA (usually 4-6 bp sequence of nucleotides) and digest the DNA at this site. • Produce fragments of DNA of various lengths. Restriction Fragments. • Restriction Enzymes isolated from many species of bacteria.
Restriction Enzymes • Several hundred isolated. • Specifically named: Ex: EcoRI E = genus co = species coli R = strain RY13 I = first endonuclease isolated
Some enzymes... • Others cleave each strand off-center at specific nucleotides to produce “overhangs”-sticky end cutters. Ex. EcoRI • Cut cleanly through the DNA at the same position on both strands- blunt end cutters. Ex. Hae III
How Do You Separate DNA Fragments? • Electrophoresis- the separation of DNA fragments based upon the negative charge of the DNA and its size. • DNA is negatively charged because of phosphates.
Genetic Variation • Any two humans have nearly identical sequences of DNA (99.9%). • But 0.1% is = to 3.0 X 106 nucleotides (recall the genome is 3.0 X 109 nucletotides). • DNA profiling takes advantage of polymorphisms (many forms) of repeated sequences in noncoding regions of DNA (near centromeres or ends of chromosomes)
Two Kinds of Polymorphisms • Variable Number Tandem Repeats (VNTR’s) – 9-80 nucleotides that repeat next to each other; the number of times they repeat varies. • Short Tandem Repeats (STR’s) – 2-8 nucleotides that repeat; also variable in the number of repeats. • VNTR and STR patterns are inherited from each parent; one set from each parent. Can be used in paternity testing, crimes scene identification, conservation biology, etc.
What’s the point? • Use different restriction enzymes to make a restriction map- relative positions of sites cut by the enzymes. • Allows you to recombine DNAs of different organisms-recombinant DNA technology. • Allows you to identify and match DNAs; DNA Fingerprinting.
Restriction Enzymes Identify Genetic Disease • Mutations in a gene change the length of the DNA fragments cut by restriction enzymes. • These different lengths are called Restriction Fragment Length Polymorphisms (RFLPs). • Fragments can be separated by electrophoresis.
An Application • Glow-in-the-Dark Pets? • http://www.msnbc.msn.com/id/30463427/