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Published by redoctober24

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Published by: redoctober24 on Mar 13, 2009
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2.40 Analysis of DNA

The techniques of DNA analysis have undergone a revolution in the past two decades. This
revolution has enabled the complete sequencing of several bacterial genomes and detailed
investigations into the regulation of growth and differentiation. These tools will play an
increasingly important role in medical care (diagnosis) and the legal system (DNA fingerprinting) in
the future.

2.41 DNA Sequencing

The most commonly used DNA sequencing method today is Sanger or dideoxy sequencing. Four
reaction mixtures are set up with all dNTPs, a DNA polymerase, radioactive dATP (to label the
DNA), a oligonucleotide primer, and the DNA to be sequenced. A small amount of one dideoxy
nucleotide triphosphate (ddNTP) is included into each reaction mixture to produce a ddATP,
ddTTP, ddGTP, and a ddCTP reaction. Then the mixture is incubated at 40 degrees C for a few
minutes and the samples are then analyzed by gel electrophoresis. Because the dideoxy nucleotide
lacks a 3\u2019-OH group, it halts DNA synthesis whenever it is incorporated into the DNA. This
produces a series of DNA fragments that end with the ddNTP that was added to that specific
reaction. When all four reaction mixtures are electrophoresed, it is possible to determine the
sequence of 100-400 basepairs of the target DNA by \u2018reading up the gel\u2019. By independently running
a separate set of reactions with a primer specific for the complimentary strand, one could identify
any errors in sequencing made with the original primer.

2.42 Restriction Endonucleases

Restriction enzymes recognize short palindromic sequences, often 4-6 basepairs in length, of double
stranded DNA and cleave a specific phosphodiester bond on each strand of the helix. The DNA
fragments produced by restriction enzyme digestion can then be analyzed by electrophoresis, used in
other molecular biology applications, and used as genetic markers. These enzymes have been
isolated from bacteria that employ these enzymes to combat bacteriophages. The target sequences
of the restriction enzymes that a bacterium possess are methylated, preventing the enzyme from
recognizing the target. Invading bacteriophage DNA will not have the target sites methylated, so the
DNA will be cleaved into small fragments and later degraded to prevent infection. Below is an
example of EcoRI, a restriction enzyme with a recognition sequence of GAATTC cutting a DNA
molecule into two smaller fragments.

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