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Analysing molecules (DNA)(BOOK)
Chapter 8 pp 463-479 top, 482-485
Analysing and manipulating DNA
Recombinant DNA technology is the ability to manipulate DNA which has had a dramatic impact on
all aspects of cell and molecular biology. Central to the technology are the following manipulations:
- Cleavage of DNA at specific sites
- DNA ligation
- DNA cloning
- Nucleic acid hybridisation
- DNA synthesis
- Determination of the sequence of nucleotides.
Cleavage of DNA at specific sites
Restriction nucleases are enzymes purified from bacteria which cut the double helix at specific sites
defined by the local nucleotide sequence. By doing so, they cleave a long double-stranded DNA
molecule into specific fragments. Restriction nucleases are called this way, because they were found
in bacteria and these bacterial nucleases restricted the transfer of foreign DNA into bacteria.
Different bacterial species produce different restriction nucleases, each cutting at a different, specific
nucleotide sequence. Knowing this can be very useful for research.
Gel electrophoresis can be used to separate DNA molecules of different sizes. Gel electrophoresis is
a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their
fragments, based on their size and charge. Because each nucleotide in a nucleic acid molecule does
already carries a single negative charge on the phosphate group there is no need to add a negatively
charged detergent. Small DNA fragments will move faster than larger ones because of the resistance
of the gel matrix. So in the end, DNA fragments spread out, sorted on fragment length along the gel
matrix.
There are different substances for the gel matrix used, depending on the size of the fragments.
Larger fragments (>500n) can be separated in a solution of agarose and smaller (<500n) in a solution
of polyacrylamide gel.
Ordinary gel electrophoresis fails to separate very large DNA molecules because the molecule will
spread out like a worm through the gel. In pulsed-field gel electrophoresis, this can be done cause
the direction of the field changes periodically. DNA bands are invisible in the gel, that is why it is
labelled/stained (radioisotopes, fluorescent, chemical marker, etc.)
DNA cloning and ligation
DNA cloning means both the act of making identical copies of a DNA molecule and the isolation of
particular stretch of DNA from the rest of the cells genome. The term has two meaning because the
isolation is usually accomplished by making identical copies of only one DNA.
Now, we only refer to the first meaning. There are multiple ways of cloning DNA. One way is by
inserting a fragment of DNA into the purified DNA genome of a self-replicating genetic element
which are usually a plasmids called plasmid vectors. For use as cloning vectors, the purified plasmide
DNA circles are first cut with a restriction nuclease to create linear DNA molecules. The DNA which
has to be cloned is added to the cut plasmid and then covalently joined by DNA ligase. These DNA
circles are then introduced back in bacterial cells. A useful plasmid vector is based on the F plasmid in
E. coli. The derivativation of it is the bacterial artificial chromosome (BAC).
, The collection of cloned plasmid molecules is known as a DNA library. The collection of DNA
fragments from the organism of interest is called a genomic library.
Another way of cloning is to begin cloning by selecting only those DNA sequences that are
transcribed into mRNA. This is done by extracting the mRNA from cells and then making a DNA copy
of each mRNA, which is called a complementary DNA or cDNA. The copying reaction is catalysed by
reverse transcriptase enzymes, which synthesis a DNA chain on an RNA template. The single stranded
cDNA molecules produced are converted by DNA polymerase into double stranded molecules and
inserted into a vector. The cloned DNA is called a cDNA clone and the entire collection of clones
derived from one mRNA constitutes a cDNA library.
Genomic libraries are especially useful in determining the nucleotide sequences of a whole genome.
The most important advantage of cDNA clones over genomic clones is that they contain the
uninterrupted coding sequence of a gene.
Although using bacteria for DNA cloning is still widely used today, there is an even simpler way of
cloning DNA which can be carried out entirely in vitro (outside an organism). This approach is called
the polymerase chain reaction (PCR).
Nucleic acid hybridisation and DNA synthesis
DNA denaturation means that the bonds between complementary base pairs are broken while the
covalent bonds between nucleotides in a strand are still intact. This can be done by heating the DNA
(around 90 degrees C). When the temperature will decline after the heating, the strands will come
back together and reform the bonds, this is called DNA renaturation or DNA hybridization.
This capacity is a powerful and sensitive technique for detecting specific nucleotide sequences. By
designing a short, single-stranded DNA molecule (DNA probe) that is complementary to the
sequence of interest a double helix can be formed.
PCR depends on both the selectivity of DNA hybridisation and the ability of DNA polymerase to copy
a DNA template. The primer needed are designed by the experimenter.
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