DNA sequencing

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Ministry of Agriculture National Research Center
for Agriculture and LivestockVeterinary
Diagnostic Laboratory -RiyadhUnit Genome

• Presentation about Seminar for
• DNA Sequencing
• Presented by Mohammed Elnour Abbo MSCs
  GenteticsMolecular Biology 2014

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What is DNA sequencing ?

• DNA sequencing is the process of
  determining the precise order of nucleotides withi
  n a DNA molecule. It includes any method or
  technology that is used to determine the order of
  the four basesadenine, guanine, cytosine,
  and thymine

On the other words the method developed by Fred
Sanger forms the basis of automated "cycle"
sequencing reactions today. Scaling up to
sequence. In the 1980s, two key developments
allowed researchers to believe that sequencing
the entire genome could be possible. The first
was a technique called polymerase chain reaction
(PCR) that enabled many copies of DNA sequence to
be quickly and accurately produced. The second,
an automated method of DNA sequencing, built upon
the chemistry of PCR and the sequencing process
developed by Frederick Sanger in 1977
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History of DNA sequencing
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History of DNA sequencing
MC chapter 12
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Method

• 1- first piece of chromosome taken contains
  millions of nucleotides this nucleotides
  converted into smaller pieces
• 2- the pieces are used as templates for
  processing it produce set of fragments of
  different length
• 3-the technique of agarose DNA gel
  electrophoresis used to separate fragments and
  keep them in order.
• 4-flourscent dyes used to separate the set of
  fragments on the agarose and keep them in
  sequence.

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• 5-the final basis at end of each strand fragment
  are identified witch when combined together make
  the short strand witch was generated in the first
  step
• 6-automated sequencers are used for analysis of
  resulting Electropherograms they show four color
  chromatogram in witch peak are showing and
  represent the four DNA bases.
• 7-computers are use and all of four fragments are
  combined together and make long stretch analyzed
  for gene coding regions and other characteristics
  .

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Basic Methods of sequencing

1. Sanger dideoxy (primer extension/chain-termination
   ) method most popular protocol for sequencing,
   very adaptable, scalable to large sequencing
   projects
2. Maxam-Gilbert chemical cleavage method DNA is
   labelled and then chemically cleaved in a
   sequence-dependent manner. This method is not
   easily scaled and is rather tedious
3. Pyrosequencing measuring chain extension by
   pyrophosphate monitoring

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Maxam-Gilbert chemical method

• Formic Acid
• Piperin
• Hydrazin
• Dimethyl sulphate

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• Sanger sequencing (Dideoxy)
• Single stranded DNA template
• A primer for DNA synthesis
• DNA polymerase
• Deoxynucleoside triphosphates and
  dideoxynucleotide triphosphates

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10_07_1_enzym.dideoxy.jpg
(B)
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How to visualize DNA fragments?

• Radioactivity
• Radiolabeled primers (kinase with 32P)
• Radiolabelled dNTPs (gamma 35S or 32P)

• Fluorescence
• ddNTPs chemically synthesized to contain fluors
• Analysis of sequencing products
• Polyacrylamide gel electrophoresis--good
  resolution of fragments differing by a single
  dNTP
• Slab gels
• Capillary gels

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Sanger Sequencer
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An automated sequencer
The output
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Future Research

• Next generation sequencing (NGS)
• Next-generation sequencing (NGS), also known as
  high-throughput sequencing, is the catch-all term
  used to describe a number of different modern
  sequencing technologies including
• Massively parallel signature sequencing (MPSS)
• Polony sequencing
• 454 pyrosequencing
• Illumina (Solexa) sequencing
• SOLiD sequencing
• Ion Torrent semiconductor sequencing
• DNA nanoball sequencing
• Heliscope single molecule sequencing
• Single molecule real time (SMRT) sequencing
• Nanopore DNA sequencing

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07_03.jpg
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IIIumina MiSeq Sequencer

• Application
• small genome sequencing,
• targeted gene expression analysis

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3500 Genetic Analyzer

• Application
• Large genome sequencing
• fragment analysis
• HID (human identification) analysis.

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Qiagen Pyromark Q96 Pyrosequencer

• Application
• Microbial identification
• Mutation analysis (SNP) single nucleotide
  polymorphism
• Methylation analysis
• Genetic testing

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Pyrophosphate group
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Introduction to bioinformatics

1. Making biological sense of DNA sequences
2. Online databases a brief survey
3. Database in depth NCBI
4. What is BLAST?
5. Using BLAST for sequence analysis
6. Biology workbench, etc.

www.ncbi.nlm.nih.gov www.tigr.org http//workbench
.sdsc.edu
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Theres plenty of DNA to make sense of
http//www.genomesonline.org/
(2006)
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Protein 3D Structure
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(we have this)
genome
(we want these)
DNA
transcriptome
RNA
proteome
protein
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Applications of DNA sequencing
Veterinary the study of animal infectious
diseases and are beginning to revolutionize the
way that biological and evolutionary processes
can be studied at the molecular level. Studies
have demonstrated the value of NGS technologies
for molecular characterization, ranging from
metagenomic characterization of unknown pathogens
or microbial communities to molecular
epidemiology and evolution of viral quasispecies.
Moreover, high-throughput technologies now allow
detailed studies of host-pathogen interactions at
the level of their genomes (genomics),
transcriptomes (transcriptomics), or proteomes
(proteomics). Ultimately, the interaction between
pathogen and host biological networks can be
questioned by analytically integrating these
levels (integrative OMICS and systems biology).
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•  Agriculture- DNA sequencing has played vital
  role in the field of agriculture. The mapping and
  sequencing of the whole genome of microorganisms
  has allowed the agriculturists to make them
  useful for the crops and food plants. For
  example, specific genes of bacteria have been
  used in some food plants to increase their
  resistance against insects and pests and as a
  result the productivity and nutritional value of
  the plants also increases. These plants can also
  fulfill the need of food in poor countries.
  Similarly, it has been useful in the production
  of livestock with improved quality of meat and
  milk. 

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• Forensics- DNA sequencing has been applied in
  forensics science to identify particular
  individual because every individual has unique
  sequence of his/her DNA. It is particularly used
  to identify the criminals by finding some proof
  from the crime scene in the form of hair, nail,
  skin or blood samples. DNA sequencing is also
  used to determine the paternity of the child.
  Similarly, it also identifies the endangered and
  protected species. Medicine- In medical
  research, DNA sequencing can be used to detect
  the genes which are associated with some heredity
  or acquired diseases. Scientists use different
  techniques of genetic engineering like gene
  therapy to identify the defected genes and
  replace them with the healthy ones.

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Advantage Disadvantage

• Advantage
• Enable the scientist to determine genome
  sequence.
• HGP Human genome project.
• Identifying genes responsible for causing genetic
  diseases
• Medical and pharmaceutical research.
• Disadvantage
• High Cost .
• Errors.

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Thank you
Mohammed Abbo. E-mail abbozam_at_gmail.com