BISC 101 Lecture Notes - Lecture 8: Primase, Molecular Sieve, Magnesium
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19 Jun 2015
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BISC 101 – Lecture 8 – Biotechnology
The DNA Toolbox
•Sequencing of the human genome was completed by 2007
•Sequencing of the genomes of more than 7,000 species was under way in 2010
•DNA sequencing has depended on advances in technology, starting with making
recombinant DNA
•In recombinant DNA, nucleotide sequences from two different sources, often two
species, are combined in vitro into the same DNA molecule
•In vitro: Biotechnology performed in a lab rather than in a living organism
DNA Cloning Yields Multiple Copies of a Gene or Other DNA Segment
•DNA cloning: Making identical copies of genes with gene – sized pieces of DNA
•Most methods for cloning pieces of DNA in the laboratory share general features
oUse of bacteria and their plasmids
•Plasmids: Small circular DNA molecules that replicate separately from the
bacterial chromosome
•Cloned genes are useful for making copies of a particular gene and producing a
protein product
•Gene cloning involves using bacteria to make multiple copies of a gene
•Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted
into a bacterial cell
•Reproduction in the bacterial cell results in cloning of the plasmid including the
foreign DNA
•This results in the production of multiple copies of a single gene
Using Restriction Enzymes to Make Recombinant DNA
•Bacterial restriction enzymes cut DNA molecules at specific DNA sequences
called restrictor sites
oAlso known as restriction endonucleases
•Evolved in bacteria as a defense mechanism against attack of foreign DNA from
invading viruses
oIn bacteria, host DNA is methylated by a modification enzyme
oMethylase protects the DNA from being cut by its own restriction enzymes
•A restriction enzyme usually makes many cuts
oThus, resulting in many restriction fragments
•The most useful restriction enzymes cut DNA in a staggered way
oThis results in sticky ends that bond with complementary sticky ends of
other fragments
•DNA ligase is an enzyme that seals the bond between restriction fragments
•Restriction recognition sites are palindromic
oThe site read the same on the reverse strand as it does on the forward
strand when both are read in the same orientation