Microorganisms Genomic Profile DrNizar

							                Microorganism Genomic Profile
Genome : The total genes content of microorganism on the entire DNA
strands (nuclear or extra nuclear (Plasmid), Mitochondrium.
Gene study: structure and location (anatomy) and function (physiology).
Value of the gene study:
1- Identification and diagnosis.
2- Typing, classification and taxonomy.

Screening Procedures:
1- Species specific primers : Primer is a short chain (around 20 bases
bp) of DNA strand from 5’ to 3’.
2-Screening to isolate one particular clone from a gene library involves
using a nucleic acid probe for hybridization. The probe will bind to the
complementary sequence allowing the required clone to be identified.
3-Restriction enzymes.
4- Sequencing.
5- Bioinformatic.
Primers
         PCR            3-Agarose gel electrophoresis




                    3-4 hours




The final product                  UV visualisation
Probe
    Methods of Molecular Diagnosis: Hybridization

   1-In Situ         Fluorescence in situ
                     hybridization (FISH)   2-Line Probe
                                 analysis




HPV L gene in skin
             cells

3-Microarray: hubdreds of
oligonucleotides idetection within few
millimeters
             Restriction Endonucleases
• Enable to cut the DNA chain for cloning and gene
  extraction.
• These restriction enzymes help in protecting bacteria
  from viruses as they cut the viral DNA leading to stop
  its work. While the bacteria protect its DNA from
  these enzymes by methylation enzymes.
• Each restriction enzyme cut at specific site on the DNA
  sequence (specificity).
• Restricion enzymes are divided according to the
  nature of cutting into two groups: 1-cut the DNA chain
  straight leading to blunt ends. 2- cut at irregular
  pattern leading to Stagger or Sticky ends.
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     Palindrome, Restriction Enzyme, Sticky Ends
    CIVIC, Madam                 Sticky Ends
                               (Cohesive Ends)
      GAATTC                  G         AATTC
       GAATTC                 AATTC                  G

        EcoRI

       Straight, Blunt Ends…. EcoRV

5’….ACTGTACGAT ATCGCTA….3’
3’….TGACATGCTA TAGCGAT….5’
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                                           Juang RH (2004) BCbasics
Methods of Molecular Diagnosis: Restriction (PFGE, pulse field gel
electrophoresis. RFLP, random fragment length polymorphism)


   M   1    2   3    4   5   6    7   8      M    RFLP analysis of adenovirus
                                                  isolates using HindIII




 PFGE profile of SmaI DNA digests of eight
 VRE isolates from hospital patients
• DNA Ligase:
• DNA ligase enzymes: ligating two ends of
  DNA strand by rebinding phosphodiester
  found between 5’ PO4 and 3’OH bonds.
    5’….ACTGTACAGATCCGCTA….3’
    3’….TGACATGTCTAGGCGAT….5’

RFLP's Map : A map used to show areas of DNA
  cuts by using different restriction enzymes
  which help to study and identify certain areas
  on this DNA strand.
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                        DNA Sequencing
The two main methods of DNA sequencing are the Maxam
  and Gilbert chemical method in which end-labeled DNA is
  subjected to base-specific cleavage reactions prior to gel
  separation, and Sanger's enzmic method. The latter uses
  dideoxynucleotides as chain terminators to produce a
  ladder of molecules generated by polymerase extension of
  a primer.
RNA Sequencing: A set of four RNases that cleave 3‫׳‬to specific
  nucleotides to produce a ladder of fragments from end-
  labeled RNA, using polacrylamide gel electrophoresis
  (PAGE) analysis allowing the sequence to be read.
Sequence Databases: Newly determined DNA, RNA and
protein sequences are entered into databases (EMBL and
GenBank). These collections of all known sequence for the
presence of patterns (eg. Restriction enzyme sites) or
similarities (eg. To new strain sequences).
Genome Sequencing Projects: The entire genome sequences
of several organisms have been determined (viruses,
bacteria, yeast worm and fly) and those of other organisms
( plant, mouse and human) are in progress. Often a genetic
map is first produced to aid the project.
Genomics: The study of organism's genome concerns with the
number, location, overall size and organization of all the
genes needed to make up an organism. The position of
pseudo-genes will aid our understanding of genome
evolution. The immediate challenge is to try to discover the
function of the huge numbers of unknown genes predicted
by genome sequencing projects. This will require large scale
gene inactivation method i.e functional genomics, coupled
with proteomic approach.
Further advances in automated DNA sequencing may use
DNA chips which are high density arrays of different DNA
sequences on a solid support as glass or nylon.
Methods of Molecular Diagnosis: Sequencing
Methods of Molecular Diagnosis: Sequencing


     Sequencing of PCR-Amplified Segment of the UL97 CMV
                            Gene to Show Point Mutations
DNA Libraries
Libraries made from genomic DNA are called genomic libraries and •
those made from complementary DNA are known as cDNA libraries.
The latter lack nontranscribed genomic sequences (repetitive
sequences,etc) Good gene libraries are representative of the starting
material and have not lost certain sequences due to cloning artifacts.
Size of Library: A gene library must contain a certain number of •
recombinants for a high probability of it containing any particular
sequence. This value can be calculated if the genome size and the
average size of the insert in the vector are known.
Genomic DNA: For making libraries, genomic DNA usually prepared •
by protease digestion and phase extraction is fragmented randomly
by physical shearing or restriction enzyme digestion to give a size
range appropriate for the chosen vector. Often combinations of
restriction enzymes are used to partially digest the DNA.
Vectors: Plasmids, λ phage, cosmid, BAC or yeast artificial •
chromosome vectors can be used to construct genomic libraries. The
choice depending on the genome size. The upper size limit of these
vectors is about 10, 23, 45, 350 and 1000 kb respectively. The
genomic DNA fragments are ligated to the prepared vector molecules
using T4 DNA ligase.
DNA Libraries
cDNA Libraries: Genomic libraries are easier to make and contain all the
genome sequence. cDNA libraries using prokaryotic mRNA is useless
since it is very unstable in the other hand cDNA libraries using
eukaryotic mRNA is very useful because the cDNA have no introns
sequences and can thus be used to express the encoded protein in E.
coli. Since they are derived from mRNA cDNA represent the
transcribed parts of the genome (i.e the gene rather than the
nontranscribed DNA) furthermore each cell type or tissue expresses a
characteristic set of genes (which may alter after stimulation or
during development). mRNA preparation from particular tissues
usually contain some specific sequences at higher abundance eg.
Globin mRNA in erythrocytes.

Bioinformatics:
Analysis of biological data by computer using special software
programs such as NCBI (National Center for Biological
Information) in which sequence alignment is adopted
making use of DNA libraries.
   Africa    Saudi

                      Indonesia
                                                SE Asia
                                        China
            Central
              Asia




                                  USA

West                                              East


   Worldwide DNA Libraries
Phylogenetic analysis