Profile Plasmid Profile Analysis - Print.pptx

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1. Plasmid Profile Analysis Plasmid = extrachromosomal DNA Profile = outline Analysis = detailed examination Outline of detailed examination of extrachromosomal DNA The labelled diagram of pBR322 plasmid is shown in the’ figure with. ( i )Eco RI and Bam HI as restriction enzymes ( Cleave DNA, Differentiate strains ) (ii) Ori and rop genes (iii) amp (an antibiotic resistant gene)

2. WHAT ARE PLASMIDS AND TRANSPOSONS AND WHY ARE THEY IMPORTANT? A plasmid is an autonomous self-replicating extrachromosomal DNA element (113). Plasmids are not essential for normal bacterial growth. Some phage genomes and plasmids are same. both can carry antibiotic resistance or toxin genes Bacteriophage = plasmids + protein coats. Plasmids are infectious => can be transferred between bacteria of the same or different genera.

3. Functions of Plasmid… Benefit the survival of the organism antibiotic resistance and toxin Synthesis of pili Plasmids are infectious Transfer and replication, Adhesin, metabolic enzyme bacteriocin production (56, 76).

4. PRINCIPLES OF PLASMID PROFILES Particular plasmid strains usually infects humans. Non-outbreak strains ≠ environmental strain Similar plasmid profiles strains but are grossly different by antimicrobial susceptibility testing, phage typing, serotyping, or another typing system are not identical. [Limitation of Plasmid profiling] One advantage of plasmid profiles over many other typing systems is that a single set of reagents and equipment is applicable to many species of bacteria .

5. METHODOLOGY add antibiotics to the growth media to maintain plasmids carrying antibiotic resistance genes (147). Plasmid profiles are relatively easy and inexpensive to perform. Plamid isolation kit $125 ( Rs : Thousand /Sample) for 12 cultures. Fresh isolates may be more difficult to lyse, and some bacteria elaborate deoxyribonuclease (193). Generally, a single nick (breakage of a covalent bond in one of the strands) will cause a plasmid molecule to be lost during the isolation procedure.

6. Weaken the cell envelope by growing the bacteria with a cell wall synthesis inhibitor such as glycine, cycloserine , or Penicillin (44, 128). The chromosomal DNA is mechanically sheared into fragments at extraction process. Alkaline lysis and denaturation of the nucleic acid form the basis of many plasmid isolation protocols. Weakened cells by lysozyme Nucleic acids are denatured SDS - Sodium Dodecyl Sulfate Sodium hydroxide to raise the pH to about 12 to 12.5. Restriction endonucleases cleave DNA at specific sequences, can be used to provide further evidence of the similarities and differences between strains.

7. Gel Compostion Agarose = 1.2 Grams Ethydium Bromide = .5-2 Microl Distilled H 2 0 = 40ml 1x Tae Buffer From 50x Stock

8. Photograph of an ethidium bromide-stained agarose gel showing plasmid profiles. Chromosomal DNA bands (arrow), two to four plasmid DNA bands, and RNA smears (near bottom) are seen in each lane. The Illinois (IL) patient and milk isolates are all identical to the outbreak strain, but the daycare isolate is missing a plasmid band and the roast beef isolate has an additional band. Plasmid DNA