GENE ISOLATION AND SEQUENCING.pdf

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.

1. Gene
Sequencing
Submitted By:
Amritanshu Pathak
M.Sc. Biotechnology, 1
st Year

2. ＊ Content
2
ϡIntroduction
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a.
b.
ϡOrgano-Chemical Gene Synthesis Mechanism
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3. ϡDNA/Gene sequencing is the determination of base order in a DNA molecule.
ϡMethods for determining base order involve either chemical degradation or, more
commonly, enzymatic synthesis of the region that is being sequenced.
ϡAutomation of the DNA sequencing process is accelerating the progress of the Human
Genome Project.
ϡKnowledge of DNA sequence and the ability to manipulate these sequences has accelerated
the development of biotechnology and has led to the development of molecular techniques.
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4. The formation of new combinations of genetic material by the insertion of nucleic acid
produced outside the cell into a virus, bacterial plasmid or any other vector system to allow its
incorporation into a host organism in which it is capable of continued replication and
expression is termed as genetic engineering.
The techniques involved are also referred to as gene cloning, in vitro genetic manipulation or
recombinant technology. The transfer of genetic material between individuals takes place in
nature by conjugation, transduction and transformation.
There are three methods to obtaining genes-
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5. “
ϡMethods for synthesis of DNA molecules of known base sequences have improved dramatically
during 1980s and 1990s.
ϡRecently, fully automated commercial instrument called automated polynucleotide synthesizer or
gene machine is available in market which synthesizes predetermined polynucleotide sequence.
ϡTherefore, the genes can be synthesized rapidly and in high amount. For examples, a gene for
tRNA can be synthesized within a few days through gene machine.
ϡIt automatically synthesizes the short segments of single stranded DNA under the control of
microprocessor.

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ϡDevelopment of insoluble silica based support in the form of beads which provides support for
solid phase synthesis of DNA chain
ϡDevelopment of stable deoxyribonucleoside phosphoramidites as synthesis which are stable to
oxidation and hydrolysis, and ideal for DNA synthesis.
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6. ϡFour separate reservoirs containing nucleotides (A, T,
C and G) are connected with a tube to a cylinder
packed with small silica beads.
ϡThese beads provides support for assembly of DNA
molecules. Reservoirs for reagent and solvent are also
attached.
ϡThe whole procedure of adding or removing the
chemicals from the reagent reservoir in time is
controlled by microcomputer control system that’s
microprocessor.
ϡIf one desire to synthesized a short polynucleotide with
a sequence of nucleotides T, G, C the cylinder is first
filled with beads with a single ‘T’ attached.
ϡTherefore, 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.
ϡThe remaning Gs which could be 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.

7. ϡGene cloning is a common practice in molecular biology labs that is used by researchers to create
copies of a particular gene for downstream applications, such as sequencing, mutagenesis,
genotyping or heterologous expression of a protein.
ϡThe traditional technique for gene cloning involves the transfer of a DNA fragment of intetrest
from one organism to a self-replicating genetics element, such as a bacterial plasmid.
ϡThis technique is commonly used today for isolating long or unstudied genes and protein
expression.
ϡIsolation of donor DNA fragment or gene
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9. ϡThe first whole RNA sequence was published in 1965 by Robert Holley and colleagues. In
parallel, a technique for separating and detecting radiolabeled RNA fragments by a
combination of charge-based separation (electrophoresis) and chromatography—called 2D
fractionation—was developed by Frederick Sanger.
ϡIn 1976, Maxam and Gilbert developed a technique in which DNA is chemically treated to
break the chain at specific bases;
ϡFollowing electrophoresis of the cleaved DNA, the relative lengths of the fragments—and
thus the positions of specific nucleotides—can be determined and the sequence inferred.
ϡThis is considered the birth of first-generation sequencing; however, the breakthrough that
would propel DNA sequencing into the future came a year later, in 1977, with Sanger’s
chain-termination method.
ϡThe , also known as Sanger sequencing, makes use of chemical
analogs of the four nucleotides.

10. ϡAllan Maxam and Walter Gilbert published a DNA sequencing method
in 1976-77 based on chemical modification of DNA and subsequent
cleavage at specific bases.
ϡThis method allows purified samples of double-stranded DNA to be
used without further cloning.
ϡMaxam-Gilbert sequencing requires radioactive labelling at end or
end of the DNA followed by purification of the DNA fragment to be
sequenced.
Procedure-
ϡRadioactive labelling of one end (5' end or 3’ end) of the DNA
fragment to be sequenced by a kinase reaction using .
ϡCut the DNA fragment with specific restriction enzyme, resulting in
two unequal DNA fragments.
ϡDenature the double-stranded DNA to single-stranded DNA by
increasing temperature.

11. ϡCleave the DNA strand at specific
positions using chemical reactions. For
example, we can use one of the two
chemicals followed by addition of
piperdine. Dimethyl sulphate (DMS)
selectively attacks purine (A and G),
while hydrazine selectively attacks
pyrimidines (C and T). This is called
modification step.
ϡChemical treatment generates breaks at
the four nucleotide bases in the four
reaction mixtures ( , and
).
ϡReagent G:
ϡReagent A+G:
.
ϡReagent C:
ϡReagent C+T:

12. ϡThe concentration of the modifying chemicals is controlled to introduce, on an average, one modification per
DNA molecule.
ϡThus a series of labelled fragments is generated, starting from the radiolabeled end to the first "cut" site in
each molecule.
ϡAs a result, we have several differently sized DNA strands in four reaction tubes.
ϡFragments are subjected to electrophoresis in high-resolution acrylamide gels for size-based separation.
ϡTo visualize the fragments, the gel is exposed to X-ray film for autoradiography, which yields a series of dark
bands, each corresponding to a radiolabeled DNA fragment, from which the nucleotide sequence may be
inferred.
ϡIn the gel, the fragments are ordered by size and, thus, we can deduce the sequence of the DNA molecule.

13. ϡThe enzymatic method is called as Sanger Method. Sanger
sequencing method was developed by Frederick Sanger and his
colleagues in 1977. The development of this technique won
Sanger the Nobel Prize in Chemistry in 1980.
ϡFrom the 1980's to the mid - 2000's, Sanger sequencing
dominated the DNA sequencing platform, bringing successful
completion of the Human Genome Project (HGP) in 2003.
Although this technique has been replaced by next generation
sequencing methods, it is still used today for smaller-scale projects.
ϡIn order to perform the sequencing, one must first convert double
stranded DNA into single stranded DNA. This can be done by
denaturing the double stranded DNA with NaOH.

14. ϡSingle stranded DNA fragment (ssDNA template): A DNA strand to be sequenced (one of the single strands,
which was denatured using NaOH).
ϡAll four deoxyribonucleotide triphosphates: i.e. dATP, dGTP, dTTP and dCTP.
ϡDNA polymerase: Each incubation tube will also carry DNA polymerase enzyme (Sequenase) in order to
copy the DNA template by adding nucleotides to the primer as the synthesis proceeds.
ϡDideoxynucleotides (ddNTPs) are chain-elongation inhibitors of DNA polymerase, used in the Sanger
method for DNA sequencing.
ϡThe main purpose of the 3'-OH group is that it is used to form a phosphodiester bond between two
nucleotides - this allows a DNA strand to elongate.
ϡThus, four sets of chain-termination fragments, corresponding to A, C, T and G are produced in four
reaction mixtures.
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ϡThe single stranded DNA is mixed with primer and split into four reaction mixtures. Each reaction
mixture contains DNA polymerase, four deoxyribonucleotide phosphates (dNTPs) and a replication
terminator(ddNTP).
ϡEach reaction proceeds until a replication terminating nucleotide (ddNTP) is added.
ϡThe mixtures are loaded into four separate lanes of gel and the electrophoresis is used to separate the
DNA fragments.

15. ϡFragmentation and amplification: Fragment the DNA and clone the fragments into vectors.
ϡDenaturation: Denature the double stranded DNA (by heat or NaOH) into single stranded
DNA fragments.
ϡAttach the primer: A primer is a synthetic oligonucleotide, containing 17 to 24 nucleotides.
The primer binds to the DNA molecule and provides a 3' OH group, which is necessary to
initiate DNA synthesis. The 3'-OH group allows for DNA chain elongation.
ϡAdd 4 dNTPs + 1 ddNTP.
ϡFour different reaction vials are taken, each with the four standard dNTPs (dATP, dGTP,
dCTP and dTTP) and DNA polymerases. Difference among the vials is because of different
type of ddNTP. Each vial will have 1 ddNTP per 100 dNTPs.
ϡAfter the occurrence of DNA synthesis, each reaction vial will have aunique set of single-
stranded DNA molecules of varying lengths. However, all DNA molecules will have the
same primer sequence at its 5' end.
ϡFind the nucleotide sequence using gel electrophoresis: In the gel, we have varying
sequences, lined up according to size.

18. ϡNot as toxic and less radioactivity than Maxam and Gilbert method.
ϡEasier to automate - Leroy Hood and co-workers used fluorescently labelled ddNTPs and
primers for the first high-throughput DNA sequencing machine. This lowered the cost from
$100 million to $10,000 USD in 2011.
ϡHighly accurate long sequence reads of about 700 base pairs.
ϡEasier to get started. The kits that are commercially available contain reagents necessary for
sequencing - pre-aliquoted and ready to use.

19. Organo-chemical gene synthesis
All gene synthesis technologies rely on the chemical synthesis of oligonucleotides to supply the building blocks for
enzymatic assembly. The most commonly used method for oligonucleotide synthesis is the cyclical four-step
phosphoramidite synthesis method developed in the 1980s (Caruthers et al., 1983, 1987; Fig. 12.1).
1.DNA oligonucleotides are synthesized in a 3′–5′ manner by coupling acid-activated deoxynucleoside
phosphoramidites to an initial deoxynucleoside attached to a solid support (usually controlled pore glass (CPG)
or polystyrene (PS) beads) through its 3′-hydroxyl group.
2.In most commercial DNA synthesizers, the solid support matrix is packed into a flow-through column and the
reagents necessary for the phosphoramidite synthesis cycle flow by the solid support matrix.
3.The addition of each nucleotide monomer to the growing oligonucleotide chain is carried out in four steps (Fig.
12.1).
4.Deprotection: A weak acid is used to remove the dimethoxytrityl (DMT) ether group from the 5′-end of the
growing oligonucleotide chain leaving a reactive 5′-hydroxyl (-OH) for the next.
5.Coupling step; The reactive 5′-OH reacts with a tetrazole-activated monomer by simultaneous addition of the
monomer and the activator solutions.
6.Capping: any uncoupled 5′-OH groups are blocked by acylation to minimize the formation of deletion products;
and finally.
7.Oxidation: the relatively unstable phosphite triester internucleotide linkages are oxidized into more stable
phosphotriester linkages

20. •This cyclic process is repeated until the
oligonucleotide is complete.
•The oligonucleotide is then cleaved from the
solid support, and the remaining protecting
groups are removed by treatment with a strong
base such as ammonium hydroxide.
•Synthesis of oligonucleotides with lengths
< 100 nt is common place using solid phase
phosphoramidite chemistry with coupling
efficiencies that can approach 99% (Rayner et
al., 1998).
•However, during acid
deprotection, depurination side reactions limit
the quality and yields of oligonucleotides above
100 nt (Hall et al., 2009). A recent
modification of the traditional reaction
conditions using a novel detritylation process
has been reported to remedy this problem and
can lead to the improved synthesis of
oligonucleotides up to 150 nt in length.

21. GENE-
SEQUENCING
References:
•https://www.europeanpharmaceuticalreview.com.
•https://www.researchgate.net.
•NIH/NHGRI
•AMU
References:
•http://www.cs.cmu.edu
•https://people.bu.edu
•https://www.csus.edu