Method of gene isloation Gene machine Pcr Gene cloning Gene sequencing methods sanger and maxum gilbert method

1. GENE SEQUENCING
Submitted To:
Dr. Amardeep Singh
Submitted by:
Muskan Singhal
M.Sc Biotechnology
1St year (II Sem)
Department of Biotechnology
Chaudhary Charan Singh University

2. Methods for gene isolation
Gene Machine
Gene Cloning
PCR

3. Fully automated machine also known as AUTOMATED
POLYNUCLEOTIDE SYNTHESIZER.
Genes can be synthesized rapidly and in high amount.
Can automatically synthesize short segment of single
stranded Dna
Firstly used by H. G Khorana in 1972 .He synthesize
Alanine trna of yeast cell
Working Principle
Insoluble silica based support
Development of stable deoxyribonucleoside
phosphoramidites as synthons which are stable to
oxidation and hydrolysis and ideal for Dna synthesis
A seperate reservoirs of A, T, C,G connected to tube to a
cyclinder packed with small silica beads
Gene Machine

4. If one desires to synthesize a short polynucleotide with a sequence of nucleotides T,G,C,
the cylinder is first filled with beads with a single 'T' attached.
Thereafter, it is flooded with 'G' from the reservoir. The right hand side of each G is
blocked by using chemicals from the reservoir so that its attachment with any other Gs
can be prevented.
The remaining Gs which could not join with Ts are flushed from the cylinder. The other
chemicals are passed from the reagent and solvent reservoirs so that these can remove
the blocks from G which is attached with the T.
In the same way this cycle is repeated by flooding with C from reservoir into the
cylinder. Finally the sequence T.G.C is synthesized on the silica beads which is removed
chemically later on.
The mechanism of a gene machine is four separate reservoirs containing nucleotides
(A,T,C and G) are connected with a tube to a cylinder (synthesizer column) packed with
small silica beads.
These beads provide support for assembly of DNA molecules. Reservoirs for reagent
and solvent are also attached.
The whole procedure of adding or removing the chemicals from the reagent reservoir in
time is controlled by microcomputer control system i.e. microprocessor.
Mechanism

5. Isolation of donor DNA fragment or gene
Selection of suitable vector
Incorporation of donor DNA fragment into the
vector
Transformation of recombinant vector into a
suitable host cell
Isolation of recombinant host cell
Steps involved in gene cloning
Gene cloning involves separation of specific gene
or DNA fragments from a donor cell, attaching it to
small carrier molecule called vector and then
replicating this recombinant vector into a host cell.
Gene Cloning

6. PCR (Polymerase Chain Reaction) is a revolutionary
method developed by Kary Mullis in the 1980s. PCR is
based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the
offered template strand. Because DNA polymerase can
add a nucleotide only onto a preexisting 3'-OH group, it
needs a primer to which it can add the first nucleotide.
This requirement makes it possible to delineate a specific
region of template sequence that the researcher wants to
amplify. At the end of the PCR reaction, the specific
sequence will be accumulated in billions of copies
(amplicons).
PCR

7. DNA template- the sample DNA that contains the target sequence. At the beginning
of the reaction, high temperature is applied to the original double-stranded DNA
molecule to separate the strands from each other.
DNA polymerase- a type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly used of these
enzymes is TaqDNA polymerase (from Thermis aquaticus), whereasPfuDNA
polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity
when copying DNA. Although these enzymes are subtly different, they both have two
capabilities that make them suitable for PCR: 1) they can generate new strands of
DNA using a DNA template and primers, and 2) they are heat resistant.
Primers- short pieces of single-stranded DNA that are complementary to the target
sequence. The polymerase begins synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates)- single units of the bases A, T,
G, and C, which are essentially "building blocks" for new DNA strands.
RT-PCR(Reverse Transcription PCR) is PCR preceded with conversion of sample RNA
into cDNA with enzyme reverse transcriptase.
Components of PCR

8. What is Gene Sequencing?
Sequencing means to determine the primary
structure of an unbranced bipolymer.
Gene Sequencing is the technique that allows
researchers to read the genetic information found in
DNA .Sequencing involves determining the order of
bases.
First sequenced genome was X174 bacteriophage in
1977 ( 5,375 bp) by Fred Sanger.

9. Sanger's Dideoxy method
Maxum and Gilbert degradation method
Techniques for Sequencing

10. Sanger's Dideoxy Method
Regions of DNA up to about 900 base pairs in length are routinely sequenced
using a method called Sanger sequencing or the chain termination method.
Sanger sequencing was developed by the British biochemist Fred Sanger and
his colleagues in 1977.
In the Human Genome Project, Sanger sequencing was used to determine the
sequences of many relatively small fragments of human DNA.
Although genomes are now typically sequenced using other methods that are
faster and less expensive, Sanger sequencing is still in wide use for the
sequencing of individual pieces of DNA, such as fragments used in DNA
cloning or generated through polymerase chain reaction (PCR).

11. Indegredients for Sanger sequencing
Sanger sequencing involves making many copies of a target DNA region.
Its ingredients are similar to those needed for DNA replication in an
organism, or for polymerase chain reaction (PCR), which copies DNA in
vitro. They include:
A DNA polymerase enzyme
A primer, which is a short piece of single-stranded DNA that
binds to the template DNA and acts as a "starter" for the
polymerase
The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)and
dideoxy nucleotides
The template DNA to be sequenced

12. Methods of Sanger sequencing
In Sanger sequencing, a DNA primer complementary to the template DNA (the DNA to
be sequenced) is used to be a starting point for DNA synthesis.
In the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the
polymerase extends the primer by adding the complementary dNTP to the template
DNA strand.
To determine which nucleotide is incorporated into the chain of nucleotides, four
dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) labeled
with a distinct fluorescent dye are used to terminate the synthesis reaction. Compared
to dNTPs, ddNTPs has an oxygen atom removed from the ribonucleotide, hence cannot
form a link with the next nucleotide.
Following synthesis, the reaction products are loaded into four lanes of a single gel
depending on the diverse chain-terminating nucleotide and subjected to gel
electrophoresis.
According to their sizes, the sequence of the DNA is thus determined.

14. In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method
based on chemical modification of DNA and subsequent cleavage at specific bases.
The method requires radioactive labelling at one end and purification of the DNA
fragment to be sequenced.
Chemical treatment generates breaks at a small proportions of one or two of the four
nucleotide based in each of four reactions (G,A+G, C, C+T).
Thus a series of labelled fragments is generated, from the radiolabelled end to the
first ‘cut’ site in each molecule.
The fragments in the four reactions are arranged side by side in gel electrophoresis
for size separation.
To visualize the fragments, the gel is exposed to X-ray film for autoradiography,
yielding a series of dark bands each corresponding to a radiolabelled DNA fragment,
from which the sequence may be inferred.
Maxam Gilbert Sequencing

15. Maxum Gilbert
Sequencing