Name :Nayab Safdar

Roll no : 53120
Topic : DNA Barcoding
 DNA Barcoding
Introduction:
 DNA barcoding is a standardized approach to identifying plants and
animals by minimal sequences of DNA, called DNA barcodes
 A DNA barcode is a short gene sequence taken from standardized
portions of the genome, use to identify species.
In plant finding conserved region is more difficult because of high
genetic diversity.
 Uses:

 Use for identifying unknown biological materials.

 Use for trade control to prevent illegal wildlife collection.
 Use to control trade of flora and fauna.
 Use to reveal cryptic species.
 Use in forensics to link biological samples to crime scenes.
 Background:
Amplification and sequence analysis of rbcl and
matK genome regions in three divergent plant
species:
 The goals of current study were four:
 Sequencing and amplification of matk and rbcl regions in three
plant species.
 Investigation of functional annotation and functional homology
modelling of the two sequences (matk + rbcl) in three species
using BLAST
 Comparative genome analysis of the sequences in various
medicinal plants
 Research the relationship between Solanum nigrum, Euphorbia
helioscopia and Dalbergia sissoo.
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 In plants, it is more difficult to find efficient conserved regions
because of the great diversity of the genome.
 Two conserved chloroplast genomic regions:
 rbcl sequence length 2500bp
 matk sequence length 1500bp
 These regions were proposed as a barcode primer to differentiate
large group of angiosperms.
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 In this study we use three divergent plant species:
 Solanum nigrum:
 Solanum nigrum commonly known as black nightshade.
 It is medicinal plant used as tonic, as antioxidant, as anti-inflammatory.
 Euphorbia helioscopia:
 It is the species of flowering plant in spurge family.
 It is also a medicinal plant used for breathing disorders.
 Dalbergia sissoo:
 Commonly known as North Indian rosewood.
 it is a perennial tree, economically important for its value in forestry,
and horticulture.
 Methods:
Plant material:
 Three plant species (S. nigrum, E. helioscopia and D. sissoo)
were used in this study.
 All three species were grown and maintained under controlled
conditions.
DNA Extraction:
 From each plant, young leaf samples were collected and ground
in liquid nitrogen to obtain a fine powder using a sterile mortar and
pestle.
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 The DNA was extracted from plant material.
 The quality of the extracted DNA was then estimated using the
Nano drop method.
 Isolated DNA was stored at -20°C.

Amplification:
 A reaction mixture for the PCR contained the following elements:
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 Forward and Reverse primers of desire gene.
 PCR amplification of the barcode regions was performed
 The amplified PCR products were electrophoresed to check
the presence or absence of tapes.
 Gel imaging was performed using the UV gel imaging
system.
 The DNA sample used for sequencing
 The final sequencing was done using ABI DNA sequencer
following standard protocol. Each sample was done in
triplicate
 Results:

 With the two universal primers rbcl and matk we observe good
PCR amplification results in three species.
 The sequence homology of the amplified sequences was
detected using the Basic Local Alignment Tool (BLAST).
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 The length of the matk sequence was 549, 852 and 296
nucleotides, respectively, with S. nigrum, E. helioscopia and D.
sissoo.
 Similarly with rbcl, the sequence length of three species was 632,
620 and 572 nucleotides.
 With matk, the sequence homology of S. nigrum and E.
helioscopia was 94% and 95% respectively while D. sissoo
showed 0% sequence homology.
 Similarly with rbcl, sequence homology of all the three species
was 96%, 99% and 96% respectively.