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Tools of Human Molecular Genetics
ANALYSIS OF INDIVIDUAL DNA AND RNA SEQUENCES
Two fundamental obstacles to carrying out their
investigations of the molecular basis of hereditary disease:
1- obtaining a sufficient quantity of a DNA or RNA sequence
of interest to allow it to be analyzed, because each cell generally
has only two copies of a gene and some genes may be transcribed
only in a subset of tissues or only at low levels, or both, providing
only a small number of messenger RNA (mRNA) molecule.
2- purifying the sequence of interest from all the other segments
of DNA or mRNA molecules present in the cell.
technological revolution that solved both the problems
of quantity and of purification. These two complementary
technologies are molecular cloning and the polymerase
chain reaction (PCR)
Molecular Cloning
Restriction Enzymes
DNA ligase
Vectors
A vector is a DNA molecule that can replicate
autonomously in a host such as bacterial or
yeast cells.
The ability to generate any desired number of
identical copies (clones) of a particular sequence
is a product of recombinant DNA technology.
Polymerase Chain Reaction (PCR)
An alternative method to cloning for generating essentially
unlimited amounts of a sequence of interest
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METHODS OF NUCLEIC ACID ANALYSIS
Several techniques are available.
Depends on the size of the DNA molecule involved:
1- Small molecules, such as normal and
recombinant versions of plasmids and phage
chromosomes.
2- large DNA molecules contained in eukaryotic
chromosomes.
Nucleic Acid Probes
Identification Methods
- It is usually easier to identify directly the
correct recombinant DNA molecule. This
can be achieved by the important
technique of hybridization probing
Probe labelling
1- Traditionally the probe is labelled with a radioactive
nucleotide,
2- non-radioactive
Radioactive labelling methods are starting to fall out
of favour, however, partly because of the hazard to the
researcher and partly because of the problems
associated with disposal of radioactive waste. The
hybridization probe may therefore be labelled in, a
non-radioactive manner.
A number of methods have been developed:
a- Biotin
b- peroxidase
Kinds of blotting
1- Southern transfer:
Transfer of DNA bands from an agarose gel to a membrane
makes use of the technique perfected in 1975 by Professor E.M.
Southern and referred to as Southern transfer.
2- northern' transfer:
used for the transfer of RNA molecules .
3- western' transfer:
used for the transfer of protein.
- Southern transfer and hybridization can be used
to locate the position of a cloned gene, or one
isolated by PCR, within any DNA molecule for
which a restriction map has been obtained.
- Southern hybridization used to determine the exact
position of a gene within the cloned fragment.
- Also the cloned fragment may carry a number of
genes in addition to the one under study.
- Southern analysis of the recombinant DNA
molecule to locate the precise position within
the cloned DNA fragment of the gene being
sought .
Southern Blotting
Northern hybridization
Locating the position of a gene on a large
DNA molecule
-Southern transfer procedure is appropriate for most
plasmids, bacteriophages and viruses, but cannot be used
to locate genes on larger DNA molecules.
- Restriction mapping becomes very complicated with
molecules more than about 250 kb in size, as can be
appreciated by referring back to Figure 4.18.
-Imagine how much more complicated the analysis would be
if there were five times as many restriction sites.
- Other techniques must therefore be used to locate
the positions of eukaryotic genes on chromosomal DNA
molecules.
In situ hybridization to visualize the position of
a gene on a eukaryotic chromosome
The much larger molecules (>50000kb) of mammals and other higher
eukaryotes are still some way beyond the capability of the current technology. Gene location on these larger DNA molecules can ,however, be achieved
by in situ hybridization, which has the added advantage :
1- Not only identifying which chromosome a gene lies on,
2-but also providing information on the position of the gene within its
chromosome.
In situ hybridization derives from the standard light microscopy techniques used to observe chromosomes in cells
In situ hybridization provides a direct visual localization of a
cloned gene on the light microscopic image of a chromosome.
Cells are treated with a fixative, attached to a glass slide. and then
incubated with ribonuclease and sodium hydroxide to degrade RNA and
denature the DNA molecules. Base pairing between the individual: polynucleotide strands is broken down, and the chromosomes unpack to a certain
extent,. exposing segments of DNA normally enclosed within their strture
in situ hybridization with radioactively labelled probes has
been used to position a number of genes on the human
cytogenetic map.
As an alternative to radioactive labelling, a fluorescent marker can
be attached to the probe and hybridization observed directly, using
a special type of light microscope.
This technique, fluorescence in situ hybridization (FISH), is also
frequently used with probes whose normal chromosomal locations are
already known. This is particularly useful for studying cells in which
chromosomal rearrangements have occurred.
DNA sequencing -working out the
structure of a gene
Probably the most important technique available to
the molecular biologist is DNA sequencing, by
which the precise order of nucleotides in a piece of
DNA can be determined. DNA sequencing methods
have been around for 35 years, but only since the
late 1970s has rapid and efficient sequencing been
possible.
Two different techniques were developed:
1- The chain termination method by F. Sanger and A.R. Coulson in the UK,
2- the chemical degradation method by A. Maxam and W. Gilbert in the USA
- The two techniques are radically different but equally
valuable. Both allow DNA sequences of several
kilobases in length to be determined in the minimum
of time.
- The DNA sequence is now the first and most basic
type of information to be obtained about a cloned
gene.
Automated DNA sequencing