ARMS AND OLA TECHNIQUES

• Similar purpose to reverse dot: test simultaneously for the presence or the
absence of a large number of polymorphisms or of potential mutations of a
gene
• Advantage over reverse dot: saves time and consumables by skipping the
steps of deposit onto the membrane, hybridization and revelation and by
replacing them by a direct visualization of the multiplex PCR amplification
products on an electrophoresis gel
 AMPLIFICATION REFRACTORY MUTATION SYSTEM (ARMS)

• Principle: amplification of a short fragment of a gene using on one side a

‘common’ primer and, on the other side, two primers that have at their 3
terminus a base complementary to that present on the standard sequence of
the gene (standard primer), or for the base present on the mutated sequence
(mutated primer). Also, these two primers are elongated so that their length
should be different.
 AMPLIFICATION REFRACTORY MUTATION SYSTEM
(ARMS):INTERPRETATION OF RESULTS
• For an individual whose two genes are not carrying the tested mutation, the
PCR will function with the common primer and the standard primer and will
give amplimers with a fixed length visible on a gel (Figure 1.19).
• For an individual carrying two identical mutated copies, the PCR will only
function with the common and mutated primers, giving amplimers of
different lengths on the gel (the mutated primer being longer or shorter
than the standard primer).
 THE OLIGONUCLEOTIDE LIGATION ASSAY (OLA)
TECHNIQUE

• more effective than ARMS because it can test a larger number of

mutations simultaneously
• automatic reading of the length of the fragment obtained when it
passes by the reading window of an automated sequencer
THE OLIGONUCLEOTIDE LIGATION ASSAY (OLA)
TECHNIQUE: PRINCIPLE
THE OLIGONUCLEOTIDE LIGATION ASSAY (OLA)
TECHNIQUE: PRINCIPLE
THE OLIGONUCLEOTIDE LIGATION ASSAY (OLA)
TECHNIQUE: PRINCIPLE
THE OLIGONUCLEOTIDE LIGATION ASSAY (OLA)
TECHNIQUE: PRINCIPLE
 RESTRICTION FRAGMENT LENGTH
POLYMORPHISMS (RFLPS)

• a difference in homologous DNA sequences that can be detected by the

presence of fragments of different lengths after digestion of the DNA
samples in question with specific restriction endonucleases
• an RFLP probe is a labeled DNA sequence that hybridizes with one or
more fragments of the digested DNA sample after they were separated by
gel electrophoresis, thus revealing a unique blotting pattern characteristic
to a specific genotype at a specific locus.
 APPLICATION OF RFLP TO THE INDIRECT GENETIC
DIAGNOSIS OF A MONOGENIC DISEASE

• RFLPs constitute a very useful tool because of their efficiency and

reliability for the indirect diagnosis of a monogenic hereditary
disease, by analysis of the co-transmission of the mutations and
the RFLP linked with the gene
• Monogenic diseases result from modifications in a single gene
occurring in all cells of the body.
 APPLICATION OF RFLP TO THE INDIRECT GENETIC
DIAGNOSIS OF A MONOGENIC DISEASE

• Monogenic diseases are responsible for a heavy loss of life. The global
prevalence of all single gene diseases at birth is approximately 10/1000.
• Examples of Monogenic Diseases – thalassemia, sickle cell anemia,
hemophilia, cystic fibrosis, Tay Sach’s disease, Fragile X syndrome,
Huntington’s disease
 IDENTIFICATION OF GENE MUTATIONS USING PCR AND
RFLP
• Some mutations can generate or remove an RFLP site.

• These mutations can be detected by PCR amplification of the sequence of

interest containing the mutation whose presence or absence has to be
tested. After amplification, the amplimers are submitted to the action of an
endonuclease associated with the site created or removed by the mutation;
visualization on a gel of the obtained fragments will allow a direct deduction
of the presence or the absence of the mutation.
 DNA MICROARRAYS

• consist of a glass or a silicon surface divided into a very large number of quadrants, on
the surface of which are oligonucleotides capable of hybridizing with a population of
genomic DNAs or cDNAs.
• A DNA chip functions like a reverse dot but the oligoprobe density is such that
thousands of different molecules can be tested simultaneously and can even be
quantified depending on the intensity of the signal collected by the visualization
system(computer controlled confocal microscopy).
• Can perform ‘genome scans’ meaning the identification of all the possible point
mutations on the whole coding sequence of a gene