Microorganism Genomic Profil e 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

PCR3-Agarose gel electrophoresis The final productUV visualisation 3-4 hours

Probe

1-In Situ Methods of Molecular Diagnosis: Hybridization HPV L gene in skin cells Fluorescence in situ hybridization (FISH) analysis 2-Line Probe 3-Microarray: hubdreds of oligonucleotides idetection within few millimeters

19/04/ 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). Stagger or Sticky ends. 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.

19/04/ Palindrome, Restriction Enzyme, Sticky Ends GAATTC G AATTC G Sticky Ends (Cohesive Ends) EcoRI CIVIC, Madam Juang RH (2004) BCbasics 5’….ACTGTACGAT ATCGCTA….3’ 3’….TGACATGCTA TAGCGAT….5’ Straight, Blunt Ends…. EcoRV

Methods of Molecular Diagnosis: Restriction (PFGE, pulse field gel electrophoresis. RFLP, random fragment length polymorphism) M M PFGE profile of Sma I DNA digests of eight VRE isolates from hospital patients RFLP analysis of adenovirus isolates using Hind III

19/04/ DNA Ligase: DNA ligase enzymes: ligating two ends of DNA strand by rebinding phosphodiester found between 5’ PO 4 and 3’OH bonds. RFLP's MapRFLP'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. 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

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

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.

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Phylogenetic analysis