1. Restriction fragment length polymorphism (RFLP) is a technique that identifies individuals based on unique patterns of restriction enzyme cutting in DNA regions. It involves digesting DNA with restriction enzymes, separating fragments by size via gel electrophoresis, transferring DNA to membranes, and detecting specific sequences via hybridization probes. RFLP is used for genome mapping, genetic disease analysis, and DNA fingerprinting. 2. Variable number tandem repeats (VNTRs) are locations with repeated sequences that vary in number among individuals. They can be analyzed via RFLP or PCR to produce unique band patterns for individual identification useful for forensics, genetics research, and studying population diversity. 3. Short interspersed

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(Recombinant DNA Technology)
Basic Concepts molecular probes, RFLP, VNTR, SINE, LINE
1. RFLP (Restriction Fragment Length Polymorphism):-Restriction Fragment
Length Polymorphism (RFLP) is a molecular method of genetic analysis that allows
individuals to be identified based on unique patterns of restriction enzyme cutting in
specific regions of DNA.
Also referred to as RFLP Analysis, the technique takes advantage of the polymorphisms in
individual people's genetic codes. Even though all members of a species have essentially the
same genetic makeup, these slight differences account for variations in phenotype, such as
appearance or metabolism, between individuals.
Step I: Restriction digest
 Extraction of desired fragments of DNA using restriction endonuclease (RE).
 The enzyme RE has specific restriction site on the DNA, so it cut DNA into fragments.
Different size of fragments are generated along with the specific desired fragments.
Step II: Gel electrophoresis
 The digested fragment are run in polyacrylamide gel electrophoresis or Agarose gel
electrophoresis to separate the fragments on the basis of length or size or molecular
weight.
 Different size of fragments form different bands.
Step III: Denaturation
 The gel is placed in sodium hydroxide (NaOH) solution for denaturation so that single
stranded DNA are formed.
Step IV: Blotting
 The single stranded DNA obtained are transferred into charge membrane ie.
Nitrocellulose paper by the process called capillary blotting or electro-blotting.
Step V: Baking and blocking
 The nitrocellulose paper transferred with DNA is fixed by autoclaving.
 Then the membrane is blocked by using bovine serum albumin or casein to prevent
binding of labelled probe nonspecifically to the charged membrane.
Step VI: Hybridization and visualization
 The labelled RFLP probe is hybridized with DNA on the nitrocellulose paper.
 The RFLP probes are complimentary as well as labelled with radioactive isotopes so
they form color band under visualization by autoradiography.

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Application of RFLP test:
 Genome mapping: helps in analysis of unique pattern in genome for organism
identification and differentiation. It also helps in determining recombination rate in the
loci between restriction sites.
 Genetic disease analysis: After identification of gene for particular genetic or
hereditary disease, that gene can be analyzed among other family members.
 To detect mutated gene.
 DNA finger printing (forensic test): It is the basis of DNA finger printing for
paternity test, criminal identification etc.
2. Variable Number Tandem Repeat :-
A variable number tandem repeat (or VNTR) is a location in a genome where a
short nucleotide sequence is organized as a tandem repeat. These can be found on
many chromosomes, and often show variations in length (number of repeats) among
individuals. Each variant acts as an inherited allele, allowing them to be used for
personal or parental identification. Their analysis is useful
in genetics and biology research, forensics, and DNA fingerprinting.
Structure and allelic variation:
In the schematic above, the rectangular blocks represent each of the repeated DNA
sequences at a particular VNTR location. The repeats are in tandem – i.e. they are
clustered together and oriented in the same direction. Individual repeats can be
removed from (or added to) the VNTR via recombination or replication errors,
leading to alleles with different numbers of repeats. Flanking the repeats are segments
of non-repetitive sequence (shown here as thin lines), allowing the VNTR blocks to
be extracted with restriction enzymes and analyzed by RFLP, or amplified by
the polymerase chain reaction (PCR) technique and their size determined by gel
electrophoresis.
Use in genetic analysis:
VNTRs were an important source of RFLP genetic markers used in linkage
analysis (mapping) of diploid genomes. Now that many genomes have
been sequenced, VNTRs have become essential to forensic crime investigations,
via DNA fingerprinting and the CODIS database. When removed from surrounding
DNA by the PCR or RFLP methods, and their size determined by gel electrophoresis
or Southern blotting, they produce a pattern of bands unique to each individual. When
tested with a group of independent VNTR markers, the likelihood of two unrelated
individuals' having the same allelic pattern is extremely low. VNTR analysis is also
being used to study genetic diversity and breeding patterns in populations of wild or
domesticated animals. As such, VNTRs can be used to distinguish strains of bacterial
pathogens. In this microbial forensics context, such assays are usually called Multiple
Loci VNTR Analysis or MLVA.
Repetitive DNA, representing over 40% of the human genome, is arranged in a
bewildering array of patterns. Repeats were first identified by the extraction
of Satellite DNA, which does not reveal how they are organized. The use of
restriction enzymes showed that some repeat blocks were interspersed throughout the
genome. DNA sequencing later showed that other repeats are clustered at specific

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locations, with tandem repeats being more common than inverted repeats (which may
interfere with DNA replication). VNTRs are the class of clustered tandem repeats that
exhibit allelic variation in their lengths.
VNTRs are a type of minisatellite in which the size of the repeat sequence is generally ten to
one hundred base pairs. Minisatellites are a type of DNA tandem repeat sequence, meaning
that the sequences repeat one after another without other sequences or nucleotides in between
them. Minisatellites are characterized by a repeat sequence of about ten to one hundred
nucleotides, and the number of times the sequence repeats varies from about five to fifty
times. The sequences of minisatellites are larger than those of microsatellites, in which the
repeat sequence is generally two to five nucleotides. The two types of repeat sequences are
both tandem but are specified by the length of the repeat sequence. VNTRs, therefore,
because they have repeat sequences of ten to one hundred nucleotides in which every repeat
is exactly the same, are considered minisatellites. However, while all VNTRs
are minisatellites, not all minisatellites are VNTRs. VNTRs can vary in number of repeats
from individual to individual, as where some non-VNTR minisatellites have repeat sequences
that repeat the same number of times in all individuals containing the tandem repeats in the
their genetic codes.
3. Short Interspersed Nuclear Elements:-
SINEs or Short Interspersed Nuclear Elements are sequences of non-coding
DNA present at high frequencies in various eukaryotic genomes. They are a class
of retrotransposons, DNA elements that amplify themselves
throughout eukaryotic genomes, often through RNA intermediates. Short-interspersed
nuclear elements are characterized by their size and method of retrotransposition. The
literature differs on the length of the SINEs but there is a general consensus that they
often range in length from about 100 to 700 base pairs (more or less, arbitrary cut-
offs). Short-interspersed nuclear elements are transcribed by RNA polymerase
III which is known to transcribe ribosomal RNA and tRNA, two types of RNA vital
to ribosomal assembly and mRNA translation. SINEs, like tRNAs and many small-
nuclear RNAs possess an internal promoter and thus are transcribed differently than
most protein-coding genes. In other words, short-interspersed nuclear elements have
their key promoter elements within the transcribed region itself. Though transcribed
by RNA polymerase III, SINEs and other genes possessing internal promoters, recruit
different transcriptional machinery and factors than genes possessing upstream
promoters.
The RNA coded by the short-interspersed nuclear element does not code for any
protein product but is nonetheless reverse-transcribed and inserted back into an
alternate region in the genome. For this reason, short interspersed nuclear elements
are believed to have co-evolved with long interspersed nuclear element (LINEs), as
LINEs do in fact encode protein products which enable them to be reverse-
transcribed and integrated back into the genome. SINEs are believed to have co-opted
the proteins coded by LINEs which are contained in 2 reading frames. Open reading
frame 1 (ORF 1) encodes a protein which binds to RNA and acts as a chaperone to
facilitate and maintain the LINE protein-RNA complex structure. Open reading frame
2 (ORF 2) codes a protein which possesses both endonuclease and reverse
transcriptase activities. This enables the LINE mRNA to be reverse-transcribed into
DNA and integrated into the genome based on the sequence-motifs recognized by the
protein’s endonuclease domain.

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Furthermore, SINEs are known to share sequence homology with LINES which gives
a basis by which the LINE machinery can reverse transcribe and integrate SINE
transcripts. Alternately, some SINEs are believed to use a much more complex system
of integrating back into the genome; this system involves the use random double-
stranded DNA breaks (rather than the endonuclease coded by related long-
interspersed nuclear elements creating an insertion-site). These DNA breaks are
utilized to prime reverse transcriptase, ultimately integrating the SINE transcript back
into the genome.[6]
SINEs nonetheless depend on enzymes coded by other DNA
elements and are thus known as non-autonomous retrotransposons as they depend on
the machinery of LINEs, which are known as autonomous retrotransposons.
4. Long interspersed nuclear elements (LINEs):-
Long interspersed nuclear elements (LINEs) (also known as Long interspersed
nucleotide element or Long interspersed elements) are a group of non-LTR (long
terminal repeat) retrotransposons which are widespread in the genome of
many eukaryotes. They make up around 21.1% of the human genome. LINEs make
up a family of transposons, where each LINE is about 7000 base pairs long. LINEs
are transcribed into mRNA and translated into protein that acts as a reverse
transcriptase. The reverse transcriptase makes a DNA copy of the LINE RNA that can
be integrated into the genome at a new site. The only abundant LINE in humans is
LINE-1. Our genome contains an estimated 105
truncated and 4 × 103
full-length
LINE-1 elements.Due to the accumulation of random mutations, the sequence of
many LINES has degenerated to the extent that they are no longer transcribed or
translated. Comparisons of LINE DNA sequences can be used to date transposon
insertion in the genome.
LINE elements propagate by a so-called target primed reverse transcription mechanism
(TPRT), which was first described for the R2 element from the silkworm Bombyx mori.
ORF2 (and ORF1 when present) proteins primarily associate in cis with their
encoding mRNA, forming a ribonucleoprotein(RNP) complex, likely composed of two
ORF2s and an unknown number of ORF1 trimers. The complex is transported back into
the nucleus, where the ORF2 endonuclease domain opens the DNA (at TTAAAA
hexanucleotide motifs in mammals). Thus, a 3'OH group is freed for the reverse transcriptase
to prime reverse transcription of the LINE RNA transcript. Following the reverse
transcription the target strand is cleaved and the newly created cDNA is integrated
New insertions create short TSDs, and the majority of new inserts are severely 5’-truncated
(average insert size of 900pb in humans) and often inverted (Szak et al., 2002). Because they
lack their 5’UTR, most of new inserts are non functional.
Regulation of LINE activity: It has been shown that host cells regulate L1 retrotransposition
activity, for example through epigenetic silencing. For example, the RNA interference
(RNAi) mechanism of small interfering RNAs derived from L1 sequences can cause
suppression of L1retrotransposition.[25]
In plant genomes, epigenetic modification of LINEs can lead to expression changes of nearby
genes and even to phenotypic changes: In the oil palm genome, methylation of a Karma-type
LINE underlies the somaclonal, 'mantled' variant of this plant, responsible for drastic yield
loss.[26]
Human APOBEC3C mediated restriction of LINE-1 elements were reported and it is due to
the interaction between A3C with the ORF1p that affects the reverse transcriptase activity.

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