Assignment 02

BS (Hons) Biotechnology, 6th Semester

Genomics
Assignment topic:
Genome Sequencing
Submitted by:
Iqra Akbar
Submitted to:
Dr. Muhammad Husnain Siddiqui
Roll no.
16630

Government College University Faisalabad

 Genomics
Genome Sequencing:
Genome sequencing is referred as making sense to find out the order of DNA
nucleotides, or bases, in a genome the basic orders of As, Cs, Gs, and Ts that make up the DNA
of an organism. The human genome is comprised of more than 3 billion of these hereditary
letters. Today, DNA sequencing got a huge scope; the scale essential for aggressive tasks, for
example, sequencing a whole genome—is for the most part done by cutting edge machines.
Much as your eye filters a grouping of letters to peruse a sentence, these machines "read" DNA
bases sequences. At the very least, the gene sequence will show an important shortcut, helping
scientists find the genes easily and quickly. Genetic sequencing contains some clues as to what
genetics are, even though scientists are just learning to interpret these details.

Question no. 01:

Describe the chain termination sequencing and chemical degradation
method?
Chain termination method:
Sanger and coworkers developed a method to cease the DNA sequencing. This method is
also called the first era DNA technique.
The strand termination approach is also called dideoxy nucleotide sequencing because of the
usage of a special sort of dideoxy nucleotide. Dideoxy nucleotide is exclusive from
traditional dNTP has a hydrogen group in dNTP now not a hydroxyl organization.
Principle:
It utilizes 2’, 3’- dideoxy nucleotide triphosphate. They are one of a kind from dNTPs at the
3’carbon. dideoxy nucleotide mainly designed nucleotides. They have a hydrogen atom
connected to the 3’ carbon as a substitute tan an OH organization.
 Phosphodiester bonds cannot form among two adjoining nucleotides because there are no
hydroxyl agencies in dideoxy nucleotide. Each dideoxy nucleotide has label for one of a
kind shade fluorescence. The nucleotide strand cannot be in addition synthesized and is
therefore referred to as the strand termination technique.
Basic Steps in Chain termination Reaction:
 DNA Sequence for chain termination:
The DNA sequencing sequence is used as a template for a special type of PCR called
PC-chain termination PCR. Chain PCR termination works not just with standard PCR,
but with one major difference: the addition of modified nucleotides (dNTPs) called
(ddNTPs). In a further step of standard PCR, DNA polymerase adds dNTPs to the
growing DNA barrel by strengthening the formation of a phosphodiester bond between
the free 3'-OH group of the last nucleotide and the next 5′-phosphate.

 Size Separation by Gel Electrophoresis:

In the second step, the chain-terminated oligonucleotides are separated by size via gel
electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel
matrix, and an electric current is applied; DNA is negatively charged, so the
oligonucleotides will be pulled toward the positive electrode on the opposite side of the
gel. Because all DNA fragments have the same charge per unit of mass, the speed at
which the oligonucleotides move will be determined only by size. The smaller a fragment
is, the less friction it will experience as it moves through the gel, and the faster it will
move. In result, the oligonucleotides will be arranged from smallest to largest, reading the
gel from bottom to top.

 Gel analysis and determination of DNA Sequence:

The final step is simply learning how to read the gel to find the DNA insertion sequence.
Because the DNA polymerase encodes only the DNA in the 5 'to 3' direction starting at
the given start point, each signal ddNTP will correspond to a specific nucleotide in the
original sequence (eg, the shortest fragment must intersect first nucleotide from the 5
'end, the second short fragment should terminate in the second nucleotide from the 5' end,
etc.) Therefore, by reading the gel bands from the smallest to the largest, we can
determine the sequence 5 'to 3 'of the first strand of DNA.
Chemical degradation Method:
The Maxam-Gilbert sequence is a DNA sequence method developed by Allan Maxam and
Walter Gilbert in 1977-1980. This method is based on chemical modifications of DNA i;e
nucleobase degradation via chemical process and subsequent specification of the DNA backbone
at sites close to the modified nucleotides.
The Maxam-Gilbert sequence was the first widely accepted form of DNA sequencing, and,
together with the Sanger dideoxy method, represents the first generation of DNA sequencing
methods. The Maxam-Gilbert sequence is no longer in full operation, as it has been replaced by
next-generation sequencing methods.
Procedure:
 As sequence of both strands are unknown, but if we find out the sequence of one strand,
we would get to know sequence of other one also. At first, the double stranded fragment
is separated into two single strands by applying high temperature or high ph.
 Run the single stranded fragments on gel. As lighter fragment band will move further
than heavy fragment band. The band having large number of purines would be heavier.
  Take one if Fragments based from the gel. Remote the phosphate at 5` end & incorporate
radioactive Phosphate 32-PO4 enzymatically.
 Now put all radioactively labelled fragments in four tubes.
 In tube 1, increase temperature and pH by adding NaOH, that would cause fragments to
breakdown. Dimethyl sulfate will be added that would make cuts at Adenine and Guanine
positions. In tube 2, Dimethyl sulfate & dilute HCl will be added that cuts the fragments
of Adenine position. In tube 3, reagents hydrazine & piperidine are added that would cuts
the fragments at position cytosine and Thiamine. In Tube 4, Hydrazine, Piper dine &
NaCl is added that would cuts the fragments at cytosine position. After chemical
degradation we get radioactively labelled fragments from each tube.
 All of fragments from each four tubes are pour in Gel. Four wells will make gel, in 1st
wall Fragments from 1st tube is pour, in 2nd well fragments 2nd tubes & so on fragments
would separate on gel according to size. Small fragments would move further than larger
fragments. After placing radioactive film on top of gel radioactive labelled fragments
would emit a spot at their position.

L
imitations:
⦁ It requires widespread use of hazardous chemicals.
⦁ It has a complex setup / technical complexity.
Question no. 04:
Describe how a small bacterial genome can be sequenced by
shotgun method?
Shotgun sequencing:
“Shotgun sequences include splitting DNA sequences into many smaller fragments
and re-integrating the sequences by searching for regions of overlap.”
During the early 1990s there was extensive debate about whether the shotgun approach would work
in practice, many molecular biologists being of the opinion that the amount of data handling
needed to compare all the mini-sequences and identify overlaps, even with the smallest genomes,
would be beyond the capabilities of existing computer systems. These doubts were laid to rest in
1995 when the sequence of the 1830 kb genome of the bacterium Hemophilus influenza and was
published too.

Principle of Shotgun sequencing:

 In shotgun sequencing, the complete genome is fragmented and cloned into vectors to
generate a genomic library. Each fragment within the library is in part sequenced.
 The desire is that there will be some overlap with the fragments. A computer assembles
the information and produces a basic genomic series encompassing all the fragments in
one long, acknowledged series, referred to as a contig Multiple contigs are frequently
separated by means of unknown sequence.
 The unknown series can be decided by hybridization protocols the use of the ends of the
acknowledged contigs.
 Alternatively, random PCR primers designed to the regarded sequences are used in
unison to enlarge the areas between acknowledged contigs.
 The drawback to this method is that a massive range of reactions have to be achieved to
find the appropriate pair of primers.
 Several strands of the DNA sequencing reads are obtained by performing several cycles
of this fragment and sequence. The computer programs then use the overlapping layers of
the various subjects to merge in a continuous sequence.
Shotgun Sequencing of Hemophilus influenza:
 The H. influenza genome was sequenced entirely by the shotgun approach and without
recourse to any genetic or physical map information. The strategy used to obtain the
sequence. The first step was to break the genomic DNA into fragments by sonication, a
technique which uses high-frequency sound waves to make random cuts in DNA
molecules. The fragments were then electrophoresed and those in the range 1.6–2.0 kb
purified from the agarose gel and ligated into a plasmid vector.
 From the resulting library, 19 687 clones were taken at random and 28 643 sequencing
experiments carried out, the number of sequencing experiments being greater than the
number of plasmids because both ends of some inserts were sequenced. Of these
sequencing experiments, 16% were considered to be failures because they resulted in less
than 400 bp of sequence.
 The remaining 24 304 sequences gave a total of 11 631 485 bp, corresponding to six
times the length of the H. influenzae genome, this amount of redundancy being deemed
necessary to ensure complete coverage. Sequence assembly required 30 hours on a
computer with 512 Mb of RAM, and resulted in 140 lengthy contiguous sequences, each
of these sequence contigs representing a different, non-overlapping portion of the
genome.
 The next step was to join pairs of contigs by obtaining sequences from the spaces
between them first, the library was checked to see if any clones of their two sequences
were found in different conditions.
 This left 42 gaps, which may have had DNA sequences that were unstable in the colony
and therefore were missing from the library. Closing these 'virtual gaps' is configured for
the second library, which has some sort of selector. Instead of using another plasmid, in
which an unlabeled sequence may no longer function, the second library was prepared in
the bacteriophage λ vector. This new library was evaluated with 84 oligonucleotides, one
at a time, and 84 of these oligonucleotides contained sequences of unlabeled contigs.

 The second strategy to close the gap is to use primers for oligonucleotides, from the set of
84 described above, as PCR primers for Hypenzae genomic DNA. Some oligonucleotide
pairs were selected at random and those that initiated the gap were identified as to
whether or not to give a PCR product (see Figure 6.11B). The resulting PCR products
have filled the right spaces. Some of the first pairs were chosen on a logical basis.
  To show that a small genome can be quickly downloaded in the form of a shotgun has
resulted in the multiplication of a large number of completed genomes. These projects
indicate that the sequence of explosives can be established based on the production level,
each member of his team responsible for DNA preparation, execution, or analysis of the
data. This strategy resulted in the 580 kb genome of Mycoplasma genitalium being
followed by five individuals in just eight weeks , and it is now accepted that a few
months should be enough time to extract the complete sequence of any genome under of
5 Mb, even though nothing was known about the genome before the start of the project.
The strength of a rifle system is therefore its speed and power of operation when there is
no genetic or physical map.

Q# 5: What are the Various ways in which the clone conting can be
built up?
Clone contig method is the most common method for detecting eukaryotic genome sequences
and has been used with those viral genomes that had previously been mapped genetically and / or
physically. In the clone contig approach, the genome is split into up to 1.5 Mb fragments, usually
with partial restriction, and this is cloned into a high-affinity column such as BAC or YAC.
Clone cone is formed by identifying clones that contain scattered fragments, which are
individually arranged in the form of a shotgun. Ideally the composite fragments are mapped to a
genetic and / or genetic map, so that sequence data from contig can be analyzed and interpreted
by looking at the features (e.g. STSs, SSLPs, genes) that are known to exist in a region.
Clone Contigs can be built on chromosome walking, but the path is difficult:
 The easiest way to build an overlapping series of DNA fragments is to start with a single
strand from the library, identify the second clone inserted in the first clone, and identify
the third fragment that extends over the second column, and so on.
 This is the basis of chromosome movement/walking, which was the first method
designed for the assembly of clone contigs.
 Chromosome walking was initially used to travel short distances through DNA
molecules, using Clone-based libraries made of λ or cosmid vectors.
 The most straightforward method is to use DNA insertion from the original Clone as a
purification method to evaluate all other clones in the library. Clone-embedded clones
provide excellent integration signals, and entries can serve as new ways to keep moving.

Assembly a genome from Large clone contigs:

 Using large cloned contigs changed into the technique that was taken by using the official
authorities sponsored human genome project.
  The genome is first damaged up into big fragments which can be cloned to present a
library of overlapping pieces. These fragments are inserted into high-capability vectors
together with YACs or BACs, which can also carry numerous hundred kb of DNA.
 Each fragment is then analyzed one after the other by using shotgun sequencing, which
ends up in a whole contiguous sequence. Overall, this method yields a set of what are
effectively large “cloned contigs.”

 After sequencing the individual cloned fragments, the next problem is to identify the
overlapping regions among the clones.
 As described above for closing the gaps left after shotgun sequencing, hybridization and
PCR methods are used to identify the overlapping fragments.
 Other methods include screening the cloned contigs for similarities in restriction profiles
and repetitive elements.
 However, comparing large numbers of clones by such methods is slow and tedious when
tackling very large genomes.
 The human genome of 3×109 bp would give 10,000 cloned fragments of 300,000 bp (the
maximum size for BAC/YAC inserts)—even without the 6- to 8-fold redundancy
necessary to ensure complete coverage.

Clone Conting by PCR:

 1: Samples of each clone in row A of the first microtiter tray are mixed together and a
single PCR carried out. This is repeated for every row of every tray - 80 PCRs in all.
2: Samples of each clone in column 1 of the first microtiter tray are mixed together and a
single PCR carried out. This is repeated for every column of every tray - 120 PCRs in all.
3: Clones from well A1 of each of the ten microtiter trays are mixed together and a single
PCR carried out. This is repeated for every well - 96 PCRs in all.

⦁ Newer more rapid methods for clone contig assembly:

Even when the screening step is carried out by the combinatorial PCR approach,
chromosome walking is a slow process and it is rarely possible to assemble contigs of
more than 15–20 clones by this method.
⦁ Restriction patterns: It can be generated by digesting clones with a variety of
restriction enzymes and separating the products in an agarose gel. If two clones contain
overlapping inserts then their restriction fingerprints will have bands in common, as both
will contain fragments derived from the overlap region.
⦁ Repetitive DNA fingerprints: These can be prepared by blotting a set of restriction
fragments and carrying out Southern hybridization with probes specific for one or more
types of genome-wide repeat. As for the restriction fingerprints, overlaps are identified by
looking for two clones that have some hybridizing bands in common.
⦁ Repetitive DNA PCR, or interspersed repeat element PCR (IRE-PCR): It uses
primers that anneal within genome-wide repeats and so amplify the single-copy DNA
between two neighboring repeats. Because genome-wide repeat sequences are not evenly
spaced in a genome, the sizes of the products obtained after repetitive DNA PCR can be
used as a fingerprint in comparisons with other clones, in order to identify potential
overlaps.

⦁ STS content mapping: It is particularly useful because it can result in a clone contig
that is anchored onto a physical map of STS locations. PCRs directed at individuals' STSs
are carried out with each member of a clone library. Presuming the STS is single copy in
the genome, then all clones that give PCR products must contain overlapping inserts.