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Summary SSA2 Analysis of genetic defects
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Summary and WG answers of SSA2 Analysis of genetic defects of the course MBO (molecular biology and oncology) at Leiden University.
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SSA2 Analysis of genetic defectsChapter 8 Alberts - Analyzing and manipulating
DNA
DNA recombination is useful to insert genes into another cell or organism. The technology
has makes use of several manipulations:
1. Cleaving the DNA with nucleases that have specific restriction sites
2. Ligation of the DNA with DNA ligase
3. Cloning of the DNA so that the portion of the genome (often a single gene) is purified by
copying it many times thereby leaving all the non-copied DNA as left over
4. Nucleic acid hybridization which makes it possible to identify a specific DNA or RNA
sequence. It is very sensitive and selective because there is only binding to the
complementary sequence.
5. Synthesis of DNA, even sequences that do not exist in nature
6. Rapid sequencing
Restriction nucleases are purified from bacteria and can cut the DNA at specific sites defined
by a nucleotide sequence. They therefore cut the DNA into strictly defined pieces. The size
of the piece depends on the length of the nucleotide sequence used by the restriction
nucleases (4x) Bacteria have these to destroy all foreign DNA. Its own DNA is protected by
methylation on the sequences that are the restriction site.
With gel electrophoresis you can separate the DNA molecules based on their size. This
procedure is way easier than it is for proteins as nucleotides are negatively charged
(because of the phosphate group). Therefore, there is no need for a charged detergent like
SDS. The gel consists of a diluted solution of agarose. There is also a variation called pulse-
field gel electrophoresis which allows for separation of really large fragments. Because of the
pulses, the molecules periodically re-orient themselves. This takes way longer for the large
fragments. To visualize the DNA, the gel is oaked in ethidium bromide which is fluorescent
under UV light. A more sensitive method it to incorporate radioisotopes in the DNA. Often 32P
is used. Labeling is also possible with digoxigenin. DNA polymerase makes the DNA from 5'
to 3' so it is read from 3' to 5'.
Genes can be cloned by using
bacteria. It can be achieved in
several ways, but the most used one
is with plasmid vectors. Plasmids are
small circular parts of DNA. The
DNA is cute with restriction
, nucleases. Often there is no clean cut, but there are staggered ends which allows for
basepairing again. If then the gene of interest is also cut with restriction nucleases and leave
complementary staggered ends, it can be ligated. The plasmids are in the cytoplasm of the
bacterium and double every 30 minutes. The most used plasmid is the bacterial artificial
chromosome (BAC). These can stably carry large parts of DNA. The cloning is also very
useful to produce any protein in large amounts.
The plasmids also make it possible to make a DNA library of the entire genome. For this you
insert a recombinant plasmid at such a concentration that there will be only one per cell. The
collection of cloned plasmids is called the DNA library.
Alternatively, you can make it from mRNA and
with reverse transcriptase make a cDNA
(complementary DNA). This can also be
inserted into a plasmid and then cloned to
make a cDNA library.
Cloning can also be done without the use of
bacteria, namely with the polymerase chain
reaction (PCR). For this, you need to use DNA
primers (basically probes without label). In
step 1 there is denaturation of the DNA at 84-
98 degrees. Then there is annealing of the
primers at 72 degrees. Then there is the
extension step in which there is DNA
synthesis by the Taq polymerase. Can also
make cDNA clones by first making cDNA with
reverse transcriptase. PCR can only be
performed on DNA, not RNA. PCR is also
used to make a DNA fingerprint in forensic
research.
Previously the most commonly used method
for sequencing was dideoxy/Sanger
sequencing. Nowadays there is NGS. RNA can also be sequences, but often this is via a
cDNA. The sequencing of the entire RNA repertoire is called deep RNA sequencing or RNA-
seq.
Sequencing methods:
- Dideoxy sequencing with labeled ddNTPs. The ddNTP leads to termination of the DNA
polymerase. Then with gel electrophoresis the DNA molecules can be separated in a
capillary to read out the sequence.
- Illumina sequencing is
based on fluorophores that
are specific for each
nucleotide. It is also based
on ddNTPs, but there is
temporary termination of the
chemical group that block the
elongation can be removed.
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