Week 4-Lecture 4

DNA Analysis Part II

Presented by
Dr.Ibrahim Ashraf Abdelwahab
Lecturer of Microbiology and Immunology dept.,
Faculty of Pharmacy, Pharos University In Alexandria.
 Competencies & Key elements achieved by learning outcomes of the
Lecture:
Competency Key elements Learning Outcomes
2-3- 2-3-1 Handle, identify, and dispose 1. Distinguish between different assays used in
Handle and dispose biologicals biologicals, synthetic/natural materials, the microbiological quality control of
and synthetic/natural biotechnology-based and radio-labeled pharmaceutical products.
pharmaceutical products, and other materials/products used 2. Outline the different serological and molecular
materials/products effectively and in pharmaceutical field. methods used to identify microorganisms.
safely with respect to relevant
laws and legislations.
2-5- 2-5-3 Contribute in planning and conducting 3. Determine the appropriate serological and
Contribute in pharmaceutical research studies using appropriate molecular methods used in research studies.
research studies and clinical methodologies.
trials needed to authorize
medicinal products.
3-1-Apply the principles of 3-1-2 Apply the principles of public health 4. Recognize the range of advanced laboratory
body functions to participate and pharmaceutical microbiology to select and important molecular and serological
in improving health care and assess proper methods of infection techniques.
services using evidence- control. 5. Determine the different serological and
based data. molecular methods used to identify
microorganisms and laboratory safety measures
3-1-4 Relate etiology, epidemiology, to prevent and control the effects of industrial
pathophysiology, laboratory diagnosis, and and laboratory hazards/accidents.
clinical features of infections/diseases and
their pharmacotherapeutic approaches. 6. Identify principles of various tools and
instruments, and select the proper techniques
for synthesizing and analyzing different
biological materials.
4-2- 4-2-2 Use contemporary technologies and media 9. Compile information from a variety of sources
Effectively communicate skills. (library, electronic, and online resources) to
verbally, non-verbally and in accomplish required assignments
writing with individuals and
communities.
 II . Sequencing of PCR product
Nucleotide sequencing of PCR
products is essential to (i)
confirm definitively the
specificity of amplification, (ii)
identify genetic variants
(polymorphisms,
rearrangements, translocations,
etc.), (iii) identify
uncharacterized genes, and (iv)
map these genes within the
organization of the genome.
 Sequencing of PCR product

A. Chain termination Method ( Sanger Method )

(Dideoxynucleotides sequencing ) ddNTPs
B. Pyrosequencing
C. Shotgun sequencing
D. Clone contig
 *Chain termination method
A.*Sanger method
*Dideoxy sequencing method
Disadvantage :
• Advantage: • Can’t be applied for
➢ Relatively easy large DNA sequencing
eg genome.
➢ Can be automated than
many individual genes up
to 96 genes sequenced
stimulus in a single run.
 Cont. Chain termination method
❖Principal: single-stranded DNA molecules that differ in
length by just a single nucleotide can be separated from
one another by polyacrylamide gel electrophoresis.
❖Method :
➢ preparation of single-stranded DNA molecules.
➢ anneal a short oligonucleotide to act as a primer.
➢The strand synthesis reaction; DNA polymerase enzyme
+ larger amounts of four deoxyribonucleotide
triphosphates (dNTPs:(dATP, dCTP, dGTP, and dTTP)) ,
+small amount of each of four dideoxynucleotides
(ddNTPs:(ddATP, ddCTP, ddGTP, and ddTTP)).
➢Electrophersis
 ❑Each of these dideoxynucleotides is labeled with a
different fluorescent marker.
❑The polymerase enzyme does not discriminate
between deoxy- and dideoxynucleotides
❖single-stranded DNA template: Produced by:
➢ M13 vector phage is not ideal, as cloned DNA fragments >
3 kb are unstable in an M13 vector and can undergo
deletions and rearrangements, so it is used only for short
pieces of DNA.
➢ Plasmid vectors: do not suffer instability problems, but
must convert the double-stranded plasmid into a single-
stranded form.
Sanger Method in 7 steps
There are two possibilities:
❑denaturation with alkali or by boiling but plasmid DNA
mainly contaminated with bacterial DNA and RNA, act as
false templates or primers.
❑DNA cloned as phagemid, [a plasmid with M13 origin of
replication and which can therefore be obtained as both
double- and single-stranded DNA versions ] stable and
clones up to 10Kb.

❖Thermal cycle sequencing (Does not need single strand )

a similar way to PCR, but just one primer [so only
one of the strands of the starting molecule is copied ] and
four dideoxynucleotides.
 Cont. Chain termination method

• Notes:
➢ Many DNA polymerases have dual activity—DNA
polymerization and DNA degradation in both 5′→3′
and 3′→5′ direction
• The 5′→3′ exonuclease activity →changes the
lengths of these strands so they do not run through
the polyacrylamide gel in the appropriate order.
• The 3′→5′ activity more importantly →removes a
dideoxynucleotide !!!!
➢Soln :
I. Klenow polymerase (The nuclease activity is
contained in the first 323 amino acids of the
polypeptide removed ), But low processivity (can
only synthesize a relatively short DNA strand about
250 bp.
II. Sequenase, (a modified version of the DNA
polymerase) has high processivity (up to 750bp )
and no exonuclease activity and so is IDEAL for
chain termination sequencing
 B. Pyrosequencing
Principal: detection of pulses of chemiluminescence, like the
chain termination method. The addition of a
deoxynucleotide to the end of the growing strand is
detectable
• Deoxynucleotides added separately
Advantage :
➢ more rapid than chain termination sequencing
➢ not require electrophoresis or any other fragment
separation
➢ Being automated enables hundreds of thousands of
sequences to be obtained at once, perhaps as much as
1000 Mb in a single run.
➢ method of choice for genome projects.
 Massively parallel pyrosequencing
• A higher version of pyrosequencing usually begins
with genomic DNA
❖ PCR is carried out in an oil emulsion: physically
separated from all the other droplets to enable us to
obtain the individual sequences of each fragment
❖After PCR, the aqueous droplets are transferred into
wells on a plastic strip so there is one droplet hence
once PCR product per well, the pyrosequencing
reactions are carried out in each well.
Massively parallel pyrosequencing
 Genome Sequencing
➢ A single chain termination sequencing ‫>ـــــــ‬about 750
bp
➢ single pyrosequence ‫>ـــــــ‬up to 150 bp.
• BUT the total size of a fairly typical bacterial genome is
4,000,000 bp and the human genome is 3,200,000,000
bp !!!!!!!!
SO -→automated systems
• To assemble the thousands or millions of individual
sequences into a contiguous genome sequence.
• Two different strategies:
➢ shotgun approach
➢ clone contig approach
C. Shotgun approach
• First used successfully with the bacterium Haemophilus
influenzae
• **Haemophilus influenzae genome sequencing project.
➢ Genome broken randomly by sonication into fragments
(1.6–2.0 kb ) that act as the templates for the sequencing
experiments.

➢Agarose Gel Electrophoresis.

➢ fragments of the desired size purified from the gel.
➢Cloning of these fragments.
➢chain termination sequencing experiments carried out.
➢Detect overlaps between fragments by computer.
 Cont. Shotgun approach
❖Limitation :
➢ ↑genome size,↑ mistakes so the shotgun used
mainly with the smaller bacterial genomes
➢ inappropriate for eukaryotic genomes, as these have
many repeat elements (computers assume them as
overlaps )
➢ Upon random fragmentation, some nucleotides lost
gaps appear
❖For gap closure: hybridization analysis of a clone
library prepared in a vector. The library was probed
in turn with a series of oligonucleotides
 clone contig approach
❑Advantage: accurate sequencing of a large genome that contains
repetitive DNA unlike the shotgun method
❑drawback: much more work, longer time, and higher cost to
construct the overlapping series of cloned DNA fragments.
❑ Once overlapping is detected, each cloned fragment is
sequenced by the shotgun method, and the genome
sequence is built up.
❑Fragments should be long enough to minimize the total
number needed to cover the entire genome.SO high
capacity vector is needed:
➢ Cosmid vector is designed to clone large fragments of DNA and
to grow their DNA as a virus or as a plasmid.
➢ Bacterial artificial chromosome (BAC) is an engineered DNA
molecule used to clone DNA sequences in bacterial cells.
 Cont. clone contig approach
Clone contig assembly
❖ chromosome walking:
• clone is selected randomly from the library, labeled,
and used as a hybridization probe against all the
other clones in the library, repeated again and again.
This is only for a short chromosome.
Cont. clone contig approach
❖Interspersed repeat element
PCR (IRE–PCR).
• This type of PCR uses primers
designed to anneal within
repetitive DNA sequences and
direct amplification of the DNA
between adjacent repeats.
• Particular repeats are
randomly distributed in a
eukaryotic genome, with
varying distances between
them → producing different
sizes.
 Cont. clone contig approach
• If a pair of clones gives PCR products of the
same size, they must contain repeats that
are identically spaced,
❖sequence tagged site content analysis
• sequence tagged site (STS): a specific DNA
sequence that occurs at just one position in
the genome under study.
• If two clones contain the same STS → this
means they overlap.
• STS positions are used to anchor the clone
contig onto a genome map and determine
the position of the contig within a
chromosome.