MOLECULAR BIOLOGY
TECHNIQUES TO MEDICAL
MICROBIOLOGY
PCR methodology
Materials
Target DNA
Taq polymerase
Four DNA nucleotides
Tow primerS
Reaction buffer
Temperature cycles
PCR methods
1- Denaturation: doble stranded DNA
eprated into two single strands 90-95c
2- Cooling at 30-60c
3- Annealing: Primers attached
complementary region of target DNA
4- Heating at 70c
5- Extention :The primers extended to form
a new strand of DNA
Defferentversions of PCR
1-Nested PCR: Increased sensitivity and
specifity
2-Reverse transcriptase PCR: (RT-PCR)
PCR also applied to amplification of RNA
3-Amplified fragment length
polymorphism(AFLP) replaced southern
blotting
4-Inverse PCR : Amplifies unknown DNA
Detection of antimicrobial
resistance
Table 2: PCR-based nuclic acid amplification for detection of antimicrobial resistance
Organism
Methicillin-resistanct
Staphylococcus aureus
coagulase-negative
staphylococci
Vancomycin-resistanct
Enterococcus spp.
Streptococcus pneumonia
Antimicrobial resistance
and
Enterobacteriaceae-producing
extended-spectrum B-lactamase
Mycobacterium tuberculosis
Herpes simplex virus
Cytomegalovirus
HIV
VanA,vanB,vanC1,vanC2,vanC
-3
Pbp1A
SHVand TEM-Blactamse gene
sequence
KatG.inhA,ahpC
RpoB
Thymidine kinase gene
Viral phosphotransferase gene
Reverse transcriptase gene
Southern blotting
Named after Edward M. Southern whodeveloped this procedure in 1975
Allow investigators to detrmine the
molecular weight of a restriction fragment
To measure relative amounts in different
samples
To locate a particular sequence of DNA
within a complex mixture