Genetic variants

Screening
&
scanning methods
• Over the last decade, there has been tremendous growth
in the knowledge we have about the role genes play in
causing disease.
• It is estimated that the genetic base of more than 1600
diseases have been identified while even more are being
investigated.
• Screening of the genetic disorders is done in order to find
the specific genetic problem which a person is having
such as change in chromosomes, genes, or proteins
• Molecular diagnostics primarily aim to detect
genomic variants, also known as single nucleotide
polymorphisms (SNPs), which can be either
pathogenic or benign.
• Genetic screening methods designed to detect
specific genomic variant(s).
• Genetic scanning methods aim to detect every
genomic variant within the PCR-amplified fragment
under study.
 TERMINOLOGY

• GENOME: The entire complement of

genetic material in the chromosome set
of an organism, virus or organelle
• CHROMOSOME is a molecular "package“
for carrying DNA in Nucleus of the cells.
• GENE: A region of DNA that encodes
function. Or A set of segments of nucleic
acid that contains the information
necessary to produce a functional RNA
product in a controlled manner.
• ALLELE: one of multiple alternative
forms of a single gene.
 Genetic Variants

 DNA based molecular diagnostics focus on trying to find

one or more changes in the DNA sequence that is related to
the health status of the individual analysed.

 Terms like polymorphism and mutation considered

ambiguous.

 Human Genome Variation Society ( HGVS) only uses neutral

terms like variants, alteration, and change
Basic Types of Variants

 Substitution: exchanges one base for another (i.e., a

change in a single "chemical letter" such as switching an A
to a G). Such a substitution could change a codon to one
that encodes a different amino acid and cause a small
change in the protein produced.
 Deletion: part of a chromosome or
a sequence of DNA is left out during DNA
replication. Any number of nucleotides can
be deleted, from a single base to an entire
piece of chromosome.

 Duplication: a portion of a genetic material

or a chromosome is duplicated or replicated,
resulting in multiple copies of that region.
 Insertion: the addition of one or more nucleotide
base pairs into a DNA sequence. This can often
happen in microsatellite regions due to the DNA
polymerase slipping.
 Inversion: a chromosome rearrangement
in which a segment of a chromosome
is reversed end to end. An inversion occurs
when a single chromosome undergoes
breakage and rearrangement within itself

 Translocation: a chromosome breaks

and a portion of it reattaches to a
different chromosome.
 Deletion-Insertion (indel):
 Conversion: one DNA sequence replaces a homologous
sequence such that the sequences become identical after
the conversion event
 Point Mutation
 Point mutation: is a genetic variant where a single
nucleotide base is changed, inserted or deleted from a
sequence of DNA or RNA.

 Point mutations have a variety of effects on the

downstream protein.

 These effects can range from benign (e.g. synonymous

mutations) to catastrophic (e.g. frameshift mutations), with
regard to protein production, composition, and function.
 Point mutations usually take place during DNA replication, the rate of
mutation may be increased by mutagens (physical, such as radiation
from UV rays, X-rays or extreme heat, or chemical, molecules that
misplace base pairs or disrupt the helical shape of DNA).

 Categorization (according to the function):

1) Nonsense mutations: include stop-gain and start-loss.

Stop-gain is a mutation that results in a premature termination codon (a
stop was gained), which signals the end of translation. this interruption
causes the protein to be abnormally shortened.
2) Missense mutations: code for a different amino acid. A
missense mutation changes a codon so that a different
protein is created, a non-synonymous change.
a) Conservative mutations: result in an amino acid change.
however, the properties of the amino acid remain the
same.
b) Non-conservative mutations: result in an amino acid
change that has different properties than the wild type,
the protein may lose its function, which can result in a
disease in the organism. For example, sickle-cell disease
3) Silent mutations: code for the same amino acid, a silent
mutation does not affect the functioning of the protein, a
single nucleotide can change, but the new codon specifies
the same amino acid, resulting in an unmutated protein.
this type of change is called synonymous change since the
old and new codon code for the same amino acid.
Variant Detection Methods
 Categories

 Variant detection methods divided to two types:

1) Low-throughput approaches: allowing the detection of a

single or just a few genomic variants.

2) High-throughput approaches: allowing the detection of a

hundreds or millions genomic variants.
 Genetic Screening methods

 Polymerase Chain Reaction (PCR)

 Restriction Fragment Length Polymorphism (RFLP)

 Amplification Refractory Mutation System(ARMS)

 Allele- Specific Oligonucleotide Probes (ASO)

 Oligonucleotide Ligation Assay (OLAs)

Restriction Fragment Length
Polymorphism (RFLP)

 RFLP: technique that is used to detect variations

in homologous DNA sequences, in order to distinguish
individuals, populations, or species or to pinpoint the
locations of genes within a sequence.

 principle: any genomic DNA can be differentiated

according to the presence or absence of restriction enzyme
sites. Restriction enzymes recognize and cut at a particular
site.
RFLP Principle
RFLP & PCR Principle
 Amplification Refractory Mutation
System (ARMS)

 ARMS: based on the principle that a mismatch between the

3’ nucleotide of a PCR primer and the template prevents
primer extension by Taq Polymerase.

 used for analysing known point mutations in DNA and

distinguishing between normal, heterozygous, and
homozygous mutant genotypes.
ARMS Principle
 Allele- Specific
Oligonucleotide Probes (ASO)

 ASO: based on the principle that even single nucleotide

mismatch between a probe and its target can destabilize
the hybrid.
 Conditions are carefully adjusted bo be slightly below the
Tm of the ASO-DNA hybrid (high stringency). Under these
conditions ASO-DNA duplexes that are not perfectly
complementary are unstable, and will melt.
 ASO probes can be designed to be complementary and
specific for various alleles, thus providing a simple
methodology to detect any known mutation or SNP.
ASO Principle
 Oligonucleotide Ligation Assay
(OLAs)

 OLAs: the principle lies in the ability of DNA ligase to join

short oligonucleotides covalently to each other, which
indirectly reflect the genotype of the target DNA

 OLA has been used to detect a wide variety of medically

relevant mutations causing inherited diseases or mutations
contributing to a common disease such as cystic fibrosis,
and variants occurring in isolated population such as
Finnish disease heritage.
ASO Principle
CONCLUSION

 All of the previously described techniques have been

extremely popular and constitute the basis on which
several diagnostic laboratories are operating.

 The main advantages of all these approaches are a high

detection rate and specificity, and improved heterozygote
detection.

 It is noteworthy that some of these methodologies set the

standards for the development of high-throughput systems
such as microarrays.
The End