PROCESS INVOLVED IN

DNA BARCODING
TISSUE SAMPLING

Items needed for tissue sampling:

 •Ethanol/gas burner and lighter
 •Ethanol in small jars to dip the forceps tips
 •Forceps (smooth tipped, not ribbed) or bladebefore flaming
 •Sterile centrifuge tubes (usually 1.5 or 2 mL) to sterilized between samples
 •Permanent markers to label tubestissue; spatula to transfer the tissue into the receive the tissueusedforceps before
sterilization
 •Petri dish to place the forceps while not being
 •Tissue paper to wipe excess tissue off the tube – mortar, pestle and spatula need to be •For plants: Mortar and pestle to
break leaf
 •Rack for tubes
 •Gloves
DNA EXTRACTION :

 DNA extraction is the process of isolating DNA from other cellular components. The
source of DNA can vary from whole specimens to fragments of skin, muscles, feathers,
organs, gut contents, feces, seeds, pollen, and even body swabs or cells shed into the
environment

 two main steps: releasing DNA from the cell((by disrupting nuclear, organellar, and cell
membranes through lysis) , and separating DNA from the other compounds (by isolation).
 for DNA extraction, the most convenient ones in terms of duration, cost, and safety (less
use of hazardous chemicals) are silica-based
 After lysis, DNA binds to a silica membrane. in the presence of salts and under specific
pH conditions. A series of wash steps followed by centrifugation removes proteins, other
cellular macromolecules, and salts, allowing pure DNA to be eluted from the membrane
into a collection tube for subsequent sequence analysis.
Diagram : DNA EXTRACTION
DNA PRESERVATION :

 DNA extracts should be stored in a freezer.

 For short to medium-term storage (on the scale of weeks), household freezers (-20°C) can
be used.
 For long-term storage, ultra-cold freezers (-80°C) are preferred.
PCR AMPLIFICATION :

 Each PCR cycle consists of three steps:

 Denaturation: the hydrogen bonds between the two strands of the DNA helix are broken at
high temperatures, usually > 94°C, resulting in two single strands
 2.Annealing: short synthetic nucleotide sequences(primers),designed to bind to the single
DNA strand at the complementary flanking regions of the target, bind to the site under
optimized temperature conditions (usually between 45-68°C) The forward (F) primer
attaches to the complementary site on the reverse DNA strand while the reverse(R) primer
attaches to the complementary site on the forward DNA strand; DNA synthesis is initiated
at each of the two primer sites by DNA polymerase.
 3.Extension: the polymerase extends the newly synthetized DNA sequence by
incorporating the dNTPs present in the PCR mix, creating a new strand with
complementarity to the between 65°C and 75°C depending on the template DNA. The
temperature varies protocol.
GEL ELECTROPHORESIS :

 To verify the success of amplification, PCR product are loaded onto an agarose gel
immersed in a buffer. An electrical current is applied, and any amplicons plus residual
primers will migrate towards the positive electrode. The distance travelled in the gel is
determined by the run time and the size of the DNA fragment(large amplicons run slower
than short one). By incorporating a dye which binds with the DNA, the presence and
position of the PCR products can be visualized under UV light .A DNA ladder is often
used to estimate the size of the fragments in the gel to verify the target region was
amplified.
 T he Gel electrophoresis workstation should be located on a separate bench and should
be considered as a ‘contaminated area’. Ethidium bromide, still widely used for the
visualization of DNA, is a mutagen which should be treated as a hazardous chemical.
Assign separate consumables (pipettes, tips, tube racks, permanent markers, tape,
gloves),equipment (electrophoresis set-up, gel imaging system) and coats to this station.
DNA Sequencing

 Sanger sequencing was the standard approach from 1980–2010

 High throughput sequencing (HTS) platforms (Sequel, Sequel II) developed by
Pacific Biosciences (PacBio)
 Can recover reference barcodes from 10,000 specimens per run
 The Sequel platforms are currently the most cost-effective technology for
constructing a DNA barcode reference library
 Sanger sequencing remains widely used for processing smaller numbers of
specimens
Sanger sequencing method

 Sanger sequencing, also known as the “chain termination method”, is a method

for determining the nucleotide sequence of DNA. The method was developed by
two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the
name the Sanger Sequence.
 There are three main steps to Sanger sequencing.
1. DNA SEQUENCE FOR CHAIN TERMINATION PCR
2.  SIZE SEPARATION BY GEL ELECTROPHORESIS
3.  GEL ANALYSIS & DETERMINATION OF DNA SEQUENCE
DNA SEQUENCE FOR CHAIN
TERMINATION PCR
  Special type of PCR called chain-termination PCR.
  One major difference: the addition of modified nucleotides (dNTPs) called di
deoxyribonucleotides (ddNTPs)
  Low ratio of chain-terminating ddNTPs with the normal dNTPs in the PCR
reaction
 ddNTPs lack the 3'-OH group required for phosphodiester bond formation;
therefore, when DNA polymerase incorporates a ddNTP at random, extension
ceases
DNA SEQUENCE FOR CHAIN
TERMINATION PCR
 Copies of the DNA sequence of interest, terminated at a random lengths (n) by 5’-
ddNTPs
 In manual Sanger sequencing, four PCR reactions are set up, each with only a
single type of ddNTP (ddATP, ddTTP, ddGTP, and ddCTP) mixed in.
  In automatic sanger sequencing, All ddNTPs are mixed in a single reaction, and
each of the four dNTPs has a unique fluorescent label.
SIZE SEPARATION BY GEL ELECTROPHORESIS

 Separated by size via gel electrophoresis

  DNA samples are loaded into one end of a gel matrix
 an electric current is applied
  DNA is negatively charged
 pulled towards the positive electrode on the opposite side of the gel
SIZE SEPARATION BY GEL ELECTROPHORESIS

  The smaller a fragment is, the less friction, and the faster it will move
 Manual Sanger sequencing, the oligonucleotides from each of the four PCR
reactions are run in four separate lanes of a gel. This allows the user to know
which oligonucleotides correspond to each ddNTP.
 In automated Sanger sequencing, all oligonucleotides are run in a single capillary
gel electrophoresis within the sequencing machine.
3. GEL ANALYSIS & DETERMINATION
OF DNA SEQUENCE
  Reading the gel to determine the sequence of the input DNA
  Terminal ddNTP will correspond to a specific nucleotide in the original sequence
  Shortest fragment must terminate at the first nucleotide from the 5’ end, the
second-shortest fragment must terminate at the second nucleotide from the 5’ end
 By reading the gel bands from smallest to largest, we can determine the 5’ to 3’
sequence of the original DNA strand.
3. GEL ANALYSIS & DETERMINATION OF DNA
SEQUENCE

 In manual Sanger sequencing, all four lanes of the gel are read at once, moving
bottom to top, using the lane to determine the identity of the terminal ddNTP for
each band. For example, if the bottom band is found in the column corresponding
to ddGTP, then the smallest PCR fragment terminates with ddGTP, and the first
nucleotide from the 5’ end of the original sequence has a guanine (G) base.
3. GEL ANALYSIS & DETERMINATION OF DNA
SEQUENCE

 In automated Sanger sequencing, a computer reads each band of the capillary gel, in order, using
fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent
tags in each band, and a computer detects the resulting light emitted. Because each of the four
ddNTPs is tagged with a different fluorescent label, the light emitted can be directly tied to the
identity of the terminal ddNTP. The output is called a chromatogram, which shows the
fluorescent peak of each nucleotide along the length of the template DNA.
 DNA BAR CODING
PROS AND CONS

By: Maryam Hafeez

 DNA BARCODING
ADVANTAGES DISADVANTAGES
• Enables quick species identification • Requires better quality control of reference
• Increase the discovery rate of new libraries
species • Occasionally co-amplifies non-target
• Adds data to solve taxonomic organisms with non-specific primers
uncertainty • Does not provide species resolution in all
• Allows species identification with limited groups
amounts of DNA • Sometimes requires use of multiple
• Increase cost and time efficiency markers
compared to traditional identification • Needs manual agreement on marker
methods choice
 ADVANTAGES DISADVANTAGES
• Objectively identifies species with • Standardization is difficult across
scalable protocols organisms groups
• Produces references for matching • Demands resources (equipment, labs,
DNA of unknown origin such as funding)
metagenomics and eDNA • Requires curated open access databases
• Can evolve with new technology • Risk being surpassed by novel
• Assists in revealing global technologies
biodiversity
• Needs a standardized legal framework to
• May automate bio-monitoring
prevent deepening the inequality gap
• Provides molecular context to
• May lead to misinterpretation of data with
historic specimens
sufficient knowledge of taxonomic or
• Can stimulate pubic agreement with
genetic variation
genetic tools
• Need stables infrastructure and
• Increases international access to
DNA resources economic resources
• Contributes to reliable library of life • Is vulnerable to instability of digital
preservation and cyber-security
 ADVANTAGES
• Method offers several advantages like
small amount of biological samples
needed, applicability for all life stages
and differentiation among
phenotypically alike species
• The sequences were determined by
using specific primers when the so-
call universal primers failed to give
amplifications
• Food adulteration is also included in
this method
• DNA barcodes can be linked to readily
observable morphological characters
• Protection of endangered species
• Helps in identifying agricultural pests
and disease vector
• Potentially very fast
DISADVANTAGES
• May create resource
competition between barcoding
and morphological approaches
• Barcodes sequences should be
generated from type specimens,
thus rely on classical taxonomy
• There is no DNA barcode gene
• Based on mitochondrial DNA
not nuclear DNA
• May be little or no difference
between intra and interspecific
genetic variations