DNA SEQUENCING

DIDEOXY SANGER METHOD

MAXAM GILBERT METHOD
DENATURING GEL ELECTROPHORESIS
PRESENTED TO: MAM AYESHA AWAAN
DNA SEQUENCING

• DNA sequencing refers to methods for determining the order of the nucleotides bases
adenine,guanine,cytosine and thymine in a molecule of DNA.
• The first DNA sequence was obtained by academic researchers, using laboratories
methods in the early 1970s.
• By the development of dye based sequencing method with automated analysis, DNA
sequencing has become easier and faster.
DIDEOXY SANGAR METHOD

• Also known as chain termination method

• The chain termination method is the method more usually used because of its speed and
simplicity.
• The key principle of the Sanger method was the use of dideoxynucleotide triphosphates
(ddNTPs) as DNA chain terminators.
• The chain termination method requires a single-stranded DNA template, a DNA primer,a
DNA polymerase, radioactively or fluorescently labelled nucleotides,and modified
nucleotides that terminate DNA strand elongation.
The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides
(dATP, dGTP, dCTP, dTTP) and the DNA polymerase.
ddNTp
To each reaction is added only one of the four dideoxynucleotide (ddATP, ddGTP, ddCTP, ddTTP) which are the chain
terminating nucleotides, lacking a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides,
thus terminating DNA strand extension and resulting in DNA fragments of varying length.
• The newly synthesized and labelled DNA fragments are heat denatured, and separated by
size by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four
reactions run in one of the four individual lanes (lanes A, T, G, C).
• The DNA bands are then visualized by autoradiography or UV light, and the DNA
sequence can be directly read off the X-ray film or gel image.
• A dark band in a lane indicates a DNA fragment that is result of chain termination after
incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP).
• The relative position of the different bands among the four lanes are then used to read
(from bottom to top) the DNA sequence
KEY FEATURES

• Uses dideoxy nucleotides to terminate DNA synthesis.

• DNA synthesis reactions in four separate tubes
• Radioactive dATP is also included in all the tubes so the DNA products will be
radioactive.
• Yielding a series of DNA fragments whose sizes can be measured by electrophoresis.
• The last base in each of these fragments is known.
CHEMICAL CLEAVAGE METHOD (MAXAM–GILBERT METHOD)

• In 1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on
the chemical modification of DNA and subsequent cleavage at specific bases.
• DNA fragments containing up to 300 nucleotides could be sequenced using the Maxam-
Gilbert method.
• The first step in this method involved creating a single-stranded DNA substrate
carrying a radioactive label on the 5 prime end.
• Then this single stranded DNA will add to 4 different test tubes and then add different
chemicals in each of 4 tubes.
PROCEDURE

• In the first tube add Dimethyl sulphate that add methyl group to guanine or it modify
Guanine in first tube.
• In the second tube add formic acid that will break glycosidic bond between nitrogenous
base and pentose sugar.it specifically break bond between purines (Guanine and
Adenine).
• In 3rd tube we use Hydrazine that acts on pyrimidine (Thymine and Cytosine) and split
pyrimidine ring.
• In 4th tube we add Hydrazine and NACL that will act on cytosine and modify it.
• Then treat all 4 of test tubes with piperdine it will break sugar phosphate backbone of the
DNA specifically at modified nucleotides.
• Thus, a series of labelled fragments are generated from the radiolabelled end to each
molecule's first cut site.
• The fragments in the four reactions are arranged side by side in gel electrophoresis for
size-based separation. The smallest sized DNA will migrate faster and present at the
bottom of the gel, and larger ones would be towards the top. In each of the four lanes,
there would be bands of radiolabelled DNA strands ending with a specific base.
• The gel is exposed to X-ray film for autoradiography to visualise the fragments. Wherever a labelled
fragment stopped on the gel, a radioactive tag would expose the film due to autoradiography. Thus, a
series of bands will appear on the X-ray film corresponding to the positions of the bands on the gel with
radiolabelled DNA fragments
• Reading the sequence from the autoradiogram would involve interpreting the banding pattern relative to
the four chemical reactions. For example, a band in the lanes corresponding to the C-only and the C + T
reactions would be a C. If the band were present in the C + T reaction lane but not in the C-only reaction
lane, it would be a T.
• The autoradiographic bands on the film will represent the 5prime →3 prime DNA sequence when read
from bottom to top.
DISADVANTAGES OF MAXAM GILBERT METHOD

• It requires extensive use of hazardous chemicals, including radioactive material like 32P
and hydrazine, a known neurotoxin.
• It has a relatively complex set-up / technical complexity.
• Only around 200-300 bases of confirmed DNA sequence can be sequenced manually
using this method in a few days.
DENATURING GRADIENT GEL
ELECTROPHORESIS
• Denaturing Gradient Gel Electrophoresis (DGGE) is a tool that is developed to analyze
DNA.
• DGGE is used to detect changes(mutations) in the genetic code within a sample,
and can detect as little as one base pair difference between strands of DNA.
PRINCIPLE

• The principle of DGGE is to separate DNA strands, based on their actual

base composition, or the ratio of GC to AT base pairs that make up a particular segment
of DNA.
METHOD

• This is accomplished by exposing the DNA to a gradient of denaturant (urea for example) at
elevated temperatures within a polyacrylamide gel.
• As the DNA sample progresses through the gel, from a low denaturant concentration to a higher
one, it starts to melt at varying points.
• This is similar to the DNA “unzipping.” The higher the GC content of the sample, the harder it is
to melt.
• Samples with lower GC content melt more rapidly in comparison. Therefore, they progress more
slowly within the gel, thus becoming separated from the other faster moving strands of DNA.
APPLICATIONS

Mutation Analysis
• Mutations are changes of genetic material due to a physical or chemical mutagenic agent.
• This results in subtle changes in the DNA. Doing a normal gel electrophoresis is
insufficient to detect such minute changes.
• By degenerating the DNA of which mutation is suspected, when subjected to DGGE
along with a normal DNA devoid of mutation, the separated fragments will get deposited
as distinct bands. This on comparison with the normal DNA, the mutations can be
detected.
MICROBIAL COMMUNITY STUDY

• The ability of DGGE to separate individual species within a sample also enables one to
follow the progression of communities over a period of time. This application is
extremely useful for remediation studies.
• In microbial remediation studies there will be a gradual progression of microorganisms
acting on the substrate. DGGE will help to identify the specific organism acting at any
particular point of time.
ADVANTAGES OF DGGE

• DGGE eliminates the need for expensive media and thereby allows more samples to be
processed for less money.
• It allows the researcher to work with a stable, and in most cases less pathogenic, form of
the organism.
• DGGE cuts overall project costs, as well as the required time for the analysis (in many
cases from a few weeks to just a few days).
• DGGE also allows the researcher to examine which organisms are stable, and what new
organisms may be appearing.
This Photo by Unknown Author is licensed under CC BY-SA-NC