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Chapter 10

BIO206H5 Chapter Notes - Chapter 10: Restriction Enzyme, Recombinant Dna, Dna Replication

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George S Espie

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Chapter 10 – Modern Recombinant DNA Technology
In the early 1970s, it became possible, for the first time, to isolate a selected piece of DNA from the many
millions of nucleotide pairs in a typical chromosome – and to replicate, sequence, and modify this DNA
oThese modified DNA molecules can then be introduced into another organism’s genome, where
they become a functional and heritable part of that organisms genetic instructions
These technical breakthroughs dubbed recombinant DNA technology or genetic engineering have
has a dramatic impact on all aspects of cell biology
oHave advanced our understanding of the organization and evolutionary history of complex
eukaryotic genomes
oLed to the discovery of whole new classes of genes, RNAs, and proteins
oContinue to generate new ways of determining the functions of genes and proteins in living
Recombinant DNA technology has also had a profound influence on our understanding and treatment of
oUsed to produce an increasing number of pharmaceuticals
Isolating and manipulating individual genes is not a trivial matter
oUnlike a protein, a gene does not exist as a discrete entity in cells; it is a small part of a much
larger DNA molecules
The solution to the problem of how to separate a gene from a eukaryotic genome came from the
discovery of a class of bacterial enzymes known as restriction nucleases
oCut double-stranded DNA at a particular sequence
oThey can be useful to produce a reproducible set of specific DNA fragments from any genome
Restriction Nucleases Cut DNA Molecules at Specific Sites
A search for the mechanism responsible for degrading foreign DNA in bacteria revealed a novel class of
bacterial nucleases that cleave DNA at specific nucleotide sequences
The bacteria’s own DNA is protected from cleavage by chemical modification of these specific sequences
Because these enzymes function to restrict the transfer of DNA between strains of bacteria, they were
called restriction nucleases
Different bacterial species produce different restriction nucleases, each cutting at a different, specific
nucleotide sequence
oBecause these target sequences are short (generally 4-8 nucleotides), many sites of cleavage
will occur in any long DNA molecule
The reason restriction nucleases are so useful in the laboratory is that each enzyme will cut a particular
DNA molecule, at the same sites
oThus for a given sample of DNA, a particular restriction nuclease will reliably generate the same
set of DNA fragments
The size of the fragments depends on the target sequences of the restriction nucleases
Gel Electrophoresis Separates DNA Fragments of Different Sizes
After a large DNA molecule is cleaved into smaller pieces with a restriction nuclease, the DNA fragments
can be separated from one another on the basis of their length by gel electrophoresis
oA mixture of DNA fragments is loaded at one end of a slab of gel
oWhen a voltage is applied across the gel, the negatively charged DNA fragments migrate toward
the positive electrode; larger fragments will migrate more slowly
oOver several hours, the DNA fragments become spread out across the gel according to size,
forming a ladder of discrete bands, each composed of a collection of DNA molecules of identical
oTo isolate a desired DNA fragment, the small section of the gel that contains the band is excised
with a scalpel or razor blade and the DNA is then extracted
Bands of DNA in a Gel Can Be Visualized Using Fluorescent Dyes or Radioisotopes
The separated DNA bands on the gel are not by themselves visible
To see these bands, you can expose the gel to a dye that fluoresces under UV light when it is bound to
DNA – the individual bands glow bright orang (bright white in black and white photograph)
An even more sensitive method involves incorporating a radioisotope into the DNA molecules before they
are separated by electrophoresis; 32P is often used
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