MOLECULAR TECHNIQUES
REQUIRING SEQUENCE
INFORMATION
The genome of organisms holds very useful information
abo u t taxo n o my, ph ylo gen i es , bi o geo g c raph y, an d
po pu lati o n dyn ami c s, w i th great i mpac t i n bo th th e
theoretical and practical fields of biology.
Molecular characterization may provide a reliable and
replicable tool, leading to the identification of an organism.
DNA molecular markers, or genetic markers, are identif iable
DNA sequences found at specif ic locations of the genome,
and transmitted to descendants (Semagn et al., 2006).
SSR (simple sequence repeats)
The microsatellites (also known as simple sequence
repeats, SSRs) are tandem repeats of short DNA
sequence moti fs. These markers have several
advantages:
i) each locus is well-defined and codominant;
ii) they are frequently polymorphic at population
level, due to dif ferences in the number of repeats;
and
iii) they are easily tested by PCR.
Note: However, sequence information is required in
order to design species-specif ic primers into the
Fig SSR variation among three plant Varieties I, II, and III with 8, 7 and 9 CTG
microsatellite repeats respectively in (A), (B) and (C). (D). Gel electrophoresis
SSR profile of the three Varieties I, II and III showing polymorphic bands due to
di erences in the number of CTG repeats among the Varieties I (8 CTG repeats
), II (7
CTG repeats) and III (9 CTG repeats).
MOLECULAR TECHNIQUES
PRODUCING BANDING PATTERNS
RAPD (random amplified
polymorphic DNA)
This technique is a random targeting PCR approach
based on short (10 bp) primers.
A possible mutation across one or more annealing
sites along DNA template will result in the absence of
the relative band, and subsequently in a dif fe rent
banding profile.
Thi s techni que allows fast, easy and cheap
comparison between a big set of samples when prior
sequence information is not available (Kjolner et al.,
2004).
Figure . RAPD variation between two plant Accessions I and II. (A). A section of
the double strand DNA of Accessions I and II are shown as two long parallel
thick
black lines. (B). Gel electrophoresis RAPD pattern of Accessions I and II showing
two bands (200 and 375 bps fragments) in Accession I but one band in
Accession II
(375 bps fragment). (C). Hypothetical banding patterns resulting from gel
electrophoresis of RAPD PCR products of ten accessions (1–10) of a plant
Single nucleotide polymorphisms
(SNPs)
SNPs are genetic polymorphisms that arise from
particular single base pair positional dif ferences in
DNA sequences among individuals of a species or
di erent species.
Compared to other genetic markers, SNPs are the
highest frequently occurring form of DNA sequence
polymorphisms in organisms (Manivannan et al., 2021).
SNPs are commonly located in coding and noncoding as well as intergenic regions of genomes at
varying abundance across each of these genomic
regions with a frequency of one SNP per every
100–300 bps of DNA (Fusari et al., 2008).
PCR-RFLP (PCR-Restriction
Fragment Length Polymorphism)
The technique consists of a digestion by a restriction
endonuclease of a specif ic amplif ied DNA segment;
the presence or the absence of a given restriction site
in the sequence will provide polymorphism between
individuals.
PCR-RFLP is a fast and reliable technique if applied
to known sequences
SSCP
Single-strand conformation polymorphism (SSCP)
analysis detects mutations based on the fact that
single-nucleotide changes in DNA sequences alter
t h e m o b i l i t y o f s i n g l e - s t ra n d e d D N A i n
nondenaturing gels.
SSCP is a simple and rapid method that is easily
reproducible for detecting mutations (deletions,
insertions and rearrangements) in PCR of amplif ied
DNA . For better results, fragment size should be
between 150-300 bp.
DNA Sequencing
Sequence Masters
 Fred Sanger, 1958
– Was originally a
protein chemist
– Made his first mark in
sequencing proteins
– Made his second mark
in sequencing RNA
 1980 dideoxy
sequencing