m The gene probes are the extremely useful

tools, probe signals are weak

m This difficulty was overcome by PCR tool
m It amplifies the DNA to 106 fold
m Discovered by Kerry Mullis
m Its an enzymatic reaction used to generate
copies of target sequence
m A series of temperatures have to be
maintained according to the DNA
m Exponential increase in the DNA
m Visualized through various methods
m Efficiency of amplification is not perfect
m el Electrophoresis
m SBYR green
At the beginning the dye molecule weakly
fluorescene.
These molecules have strong affinity to the double
stranded molecules
They bind to the dsDNA molecule and emit light
m FRET: Fuorescence Resonance Energy
Transfer
Two sequence specific oligonucleotides labeled with
fluorescent dyes
Reporter dye and quencher
m Detection by oligomer hybridisation
32P- labled probe hybridizes in solution to one strand
of the denatured amplified product
The probe target duplex is seperated from the
unhybridized probe by polyacrylamide gel
electrophoresis and visualized by autoradiography
1. Reverse Transcriptase ± 10. Colony ±PCR
PCR 11. Helicase Dependent
2. Integrated Cell Culture ± Amplification HDA
PCR 12. Hot Start ±PCR
3. Seminested, Nested and 13. Inverse ±PCR
Multiplex ±PCR 14. In Situ ±PCR
4. Fingerprinting ±PCR 15. ISSR ±PCR
5. Real Time ±PCR 16. Single Cell ±PCR
6. Amplified Fragment 17. Touchdown ±PCR
Length Polymorphism ±
PCR 18. Solid Phase ±PCR
7. Allele Specific ±PCR 19. Quantative ±PCR
8. Assembly ±PCR
9. Asymmetric ±PCR
 Heat causes DNA strands to separate

5´ 3´
3´ 5´
Denature DNA strands 94oC
5´ 3´

3´ 5´
 È rimers bind to the template sequence
È
aq olymerase recognizes double-
double-stranded substrate

5± 3±

3± 5±

rimers anneal 64oC

5± 3±
5± 3± 3± 5±
3± 5±
 È
aq olymerase e tends primer
ÈDNA is replicated
5± 3±
5± 3± 3± 5±
3± 5±

r tend 72oC

5± 3±
5± 3± 3± 5±
3± 5±

Repeat denaturing, annealing, and e tending 30 cycles

 6
6

 
6

6  

5´ 3´
3´ 5´
Cycle 1 5´ 3´
3´ 5´

3´
5´
3´ 5´
Cycle 2
5´ 3´
3´ 5´

3´ 5´
Cycle 3 5´ 3´
3´ 5´
5´ 3´
˜


 
 
m Putative insertion sequence designated as
IS6110
m Its present in high copy number
Its specific and has high repetitive nature makes it ideal
target for amplification by PCR
The internal control DNA has been constructed which
uses same primer as target sequence
Use of internal control DNA allows assessment of the
efficacy of each individual reaction ensures that the
reaction is not inhibited by interfering substances
The PCR of internal control DNA is 600bp and easily
detectable from the 123kb of the product
DNA fingerprinting of ˜

strain has been
developed using probes of IS6110




m Sample Requirements
Sputum, bronchoalveolar lavage specimen, and
bronchial washings.
m The cells are lysed
m DNA extracted
m PCR is performed
m Amplification
m Concentrated, separate and electrophoresis
m Analysis of PCR product
@


m Load the sample with bromophenol blue
indicator and stain it with ethidium
bromide on the 12% polyacrylamide gel
m Electrophoreses at 200V
m Observe under UV transilluminator
Positive: 123 bp fragment detect
Negative: 600 bp fragment detected
 Pvu II Bam HI

5´ 3
´
IR IR
Target

T4
75
759-778 881-862

123bp PCR PROCUCT

Sal I





6

m One of the two etiologic agent
m PCR is highly sensitive
m Discordance in the primer pairs include
Sequence heterogenicity
Differential analytical sensitivity of the different
primes
Short primers capable of accommodating
mismatches
Sampling bias
False positive result
m uidelines
1. Highly conserved regions to be selected and
amplified
2. Primers should be relatively long
3. To accommodate mismatches should have
3±terminal
4. Annealing temperature which will provide
maximum mismatch stability
5. dUTP and uracil-i-glycosylase should be
incorporated into the amplification reaction to
minimize false positive PCR. This also helps to
increase specificity in amplification




m Collection of blood sample

m Processing
m RT-PCR performed
m Amplification
m Concentrated, separate and electrophoresis
m Detection by oligomer hybridisation
 HIV-1 9.7kb

HIV-1 gag
791* 2299
SK462 SK431

SK102

1366 1507
123bp EXPECTED PCR PROCUCT
SK462 SK102 SK431 142 bp
(1366-1395) (1403-1435) (1507-1481)
` 




m Its region of the ribosomal DNA (rDNA) of several
isolates of `  
m Three specific primers of the fungi `  
m The primers do not amplify the sequence from
other sequences
m They have the potential to recognize the fungi in
the clinical specimens
m PCR fingerprinting: one primer is used to
distinguish between the two varieties of the fungi
m This procedure allows the isolates to be
fingerprinted
Type Name Nucleotice sequence , 5± TO 3± Location on target

C. Neoformans NS1 TA TC ATA TC TT TCT C 5±

identification
NA8 TTC CA T TCA CCT AC A 3±

eneral ITS1 TTC TA T AA CCT C A 5±

ITS4 TCC TCC CT TAT TA TAT C 3±

C. Neoformans specific CN4 ATC ACC TTC CCA CTA ACA CAT T 3±

CN5 AA  CAT CC TT TT AA  5±

CN6 TTT AA C AC CA CT CCT T 3±

PCR fingerprinting (T) Random

(ACA) Random

A T C T TCT Random





m DNA extraction
m PCR
m Preperation of primers
m Amplification
m Concentrated, separate and electrophoresis
m The result is given in the figure
 

m Its is the only species being transmitted
to the succesive genration
m It has a unique 18s rRNA which is one of
the most ancient
m It contains many species specific
regions
m Assay detects the 18s rRNA gene
sequence in fecal specimens obtained

 









 

Function Name Nucleotice sequence , 5± TO 3± Location

Upstream primer JW1 C CAC CA AA

C


1251-1270

JW2 TCA CCT AC AT ACC TT TT 1433-1414

Internal probe RDR34 A AC C TCC C  1306-1291





m The fecal specimens collected
m Sample preperation
m Extraction of the DNA
m PCR amplification
m Detection and analysis of the amplified
product
 3´
5´ G. Lamblia 18S rRNA gene

1251-1270 1251-1270
JW1 JW2

1251 1433

183bp EXPECTED PCR PROCUCT

Internal oligonucleotice
probe
13906-1291
Probe RDR34
m Detect specific nucleic
acid
m Sensitive, large target
m Independent access to
viable and nonviable
m Optimises
m Fingerprinting allows to
discriminate the products
m Access level of gene
expression
—

m Inhibition due to humic
substances and metals
m Non specific priming
m Infectious and
noninfectious mixture of
the sample
 




m Whiteley et al. # Wu and Wallace
m Target dependent ligation
m LCR employs a thermostable DNA polymerase
and thermostable DNA ligase
m It uses four oligonucleotide probes of 20-24b each
m Each pair of the oligonucleotide is designed to
bind to the denatured target DNA
m A pair of oligonucleotides are made to bind to one
of the DNA target strands
m A second pair of oligonucleotides is designed to
hybridize to the same regions on the
complementary DNA.
m The oligonucleotides are mixed with extracted
target DNA, denatured
m The reaction is then cooled allowing the
olingnucleotide probes to bind to the target DNA
m DNA polymerase fills the gap and linked by DNA
ligase yielding doublestranded DNA molecule
40-50bp length
m The cycle is repeated 30 -40 times
m The power of LCR is its compatibility with other
replication-based amplification methods.
m By combining LCR with a primary amplification,
one effectively lines up the crosshairs to
distinguish single base-pair changes with
pinpoint accuracy.
A G
T C

Denature the DNA 94°C

A G

T C

Anneal the oligonucleotides 65°C

A G
T

A
T C

Ligate with thermostable DNA ligase 65°C

A G
T

A
T G
 Repeat the cycle 20-30 times

A G
T

A
T

A
T

A
T C

R

   
primers anneal to their complementary strands at 65~ which is appro imately 5~
below their
in.
hermostable ligase (Q) will only ligate primers that are perfectly complementary to their target sequence and
hybridize directly adjacent to each other (as shown with        

! !! " target as well as on the primers for clarity. rimers that have at least a single
base-pair mismatch at the 3´ end contributing to the junction of the two primers will not ligate (as shown with L. 
 
 !#$%!! 

   & ends to avoid ligation of the 3´ ends.
 rinciple of LCR. ·  "!''    · 
   ·
!    be distinguished from other
closely related   !!·
%!difference in the 16S rDNA/2~ 
  
%! 
 ($&)'
 *%!  
! 
 
m The assay amplification kit uses four
probes complementary to a 48-bp region
of the gonococcal opacity 1 (

m ·  accession name, NOOPC
B) This gene sequence is repeated up to
11 times in the i
  

m 6   


 
i 

m
o resolve discrepant culture-negative,
m LCR-positive specimens, another LCR assay with the pilin probe set specific
for  '

m ! ! ! as described previously
(1a).
he specificity of the pilin LCR assay for  ' 
+' '
 #
 ' specimens.
m
he pilin LCR assay procedure was similar to the  
!
#"!     was for 53 cycles of
858C for 30 s and 608C for 1 min and 100 ml of each sample diluted 1:1 with
  ! 
'
m Specimens that gave duplicate IM signals above 100 counts per second per
second were confirmed as  ! #
o resolve the
results for specimens that were culture positive.
m  LCR assay negative, samples were diluted with specimen
transport buffer and were retested by the  
  'available, were also heated in specimen transport
buffer and were tested by the  

m Design of Primers
Single basepair overhangs
Temperature range
Unique primers
Noncomplementary tails
m Conditions
One set of four primers
Thermostable ligase
There is no extention
m Detection methods for product
Labeled with biotin 5±end and nonisotopic
reporter at 3± end
Dye include fluorescein dye in blue (FAM, 5-
carboxyfluorescein) and digoxigenin.
anti-digoxigenin antibodies coupled to alkaline
phosphatase (AP) greatly improved the
sensitivity.
Subsequent detection of the AP could be
achieved using colorimetric, fluorescent, or
luminogenic substrates.
m Applications
enetic , viruses and bacterial diseases
v
m Background amplification due to blunt
end ligation of probe duplexes
m Long reactions
m Carryover contaminations
m Nonstringent annealing conditions
6
v
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. All three systems can be used to form
the basis of a group of assays called
competition or inhibition ELISAs.
5. Reverse ELISA
6. ELISA spot
R

m Antigen added to solid phase, incubation
m Non bound antigens washed
m Antigen specific antibody with enzyme
conjugate added which binds antigen
m unbound antibody washed
m Substrate/ chromophore added
m Enzyme catalyzes reaction to give colour
 
v
m Antigen added to solid phase, incubation
m Non bound antigens washed
m Antigen specific antibody added which
binds antigen
m Antibodies labeled with enzyme
(conjugate) which are antispecies
m Excess is washed off
m Substrate/ chromophore added
m Enzyme catalyzes reaction to give colour
  v
 
   

 

m Antibodies, Wash m Antibody, wash

m Antigen sp, wash m Antigen sp, wash
m Antibody enzyme m Antibody sp to antigen
conjugaete m Anti-antibody enzyme
m Substrate conjugate
m Enzyme catalyzed to m Substrate
give colour m Enzyme catalyzed to
give colour


v
m Incubate antibody with antigen
m Add antigen-antibody mixture to the well coated with
antigen (The more antigen in the sample, the less
antibody will be able to bind to the antigen in the well,
hence "competition´)
m Wash
m Add enzyme conjugate secondary antibody
m Wash
m Add substrate
m Enzyme catalyzes the reaction to give colour
m For competitive ELISA, the higher the original antigen
concentration, the weaker the eventual signal.
m The major advantage of a competitive ELISA is the
ability to use crude or impure samples and still
selectively bind any antigen that may be present.
Enzymes : Chromogens /Substrates
Horse radish peroxidase
Ortho phenylene diamine/ hydrogen peroxide
2,2±azino-diethylbenzaldehyde/hydrogen peroxide
Tetra methyl benzidine/ hydrogen peroxide
5-amino salicylic acid / hydrogen peroxide
Diaminobenzidine/ hydrogen peroxide
Alkaline phosphatase
Para-nitro phenylphosphate
Ǻ-alactosidase
Ortho-nitrophenyl ȕ-galactosidase
Urease
Bromocresol purple





m Antigen coated microtitre plate
m Diluted control serum and test sample are added
m If the sample contains antibodies Ig it will bind
to the microtitre plate
m Unbound are washed out
m Enzyme conjugate: Anti human Ig peroxidase
conjugate added
m Unbound antibodies washed
m Substrate :Tetramethylbenzidine is added
m Blue coloured soluble product which turns into
yellow after adding the acid stopping solution
˜ 
 
m ELISA methodology for measurement of
Ig Ab are available.
m 38-Kd Ag provides serodiagnostic test
with most favorable test characteristics
described, but is limited by the lack of
purified Ag.
m Serum Ig Ab are observed to rise
during the first 3 months of therapy but
fall after 12-16 months.
@


v
m Advantages m Disadvantages