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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)
analysis
HPV L gene in skin
cells
3-Microarray: hubdreds of
oligonucleotides idetection within few
millimeters
2-Line Probe
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
Sticky Ends
(Cohesive Ends)
CIVIC, Madam
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)
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PFGE profile of SmaI DNA digests of eight
VRE isolates from hospital patients
M
RFLP analysis of adenovirus
isolates using HindIII
• 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 endlabeled 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
China
Central
Asia
SE Asia
USA
West
Worldwide DNA Libraries
East
Phylogenetic analysis