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The gene probes are the extremely usefultools, probe signals are weak
This difficulty was overcome by PCR tool
It amplifies the DNA to 106 fold
Discovered by Kerry Mullis Its an enzymatic reaction used to generate
copies of target sequence
A series of temperatures have to be
maintained according to the DNA Exponential increase in the DNA
Visualized through various methods Efficiency of amplification is not perfect
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Gel Electrophoresis
SBYR greeny At the beginning the dye molecule weakly
fluorescene.y These molecules have strong affinity to the double
stranded molecules
y They bind to the dsDNA molecule and emit light
FRET: Fuorescence Resonance EnergyTransfery Two sequence specific oligonucleotides labeled with
fluorescent dyes
y Reporter dye and quencher
Detection by oligomer hybridisationy 32P- labled probe hybridizes in solution to one strand
of the denatured amplified product
y The probe target duplex is seperated from theunhybridized probe by polyacrylamide gel
electrophoresis and visualized by autoradiography
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1. Reverse Transcriptase PCR
2. Integrated Cell Culture PCR
3. Seminested, Nested and
Multiplex PCR4. Fingerprinting PCR
5. Real Time PCR
6. Amplified Fragment
Length Polymorphism PCR
7. Allele Specific PCR
8. Assembly PCR
9. Asymmetric PCR
10. Colony PCR11. Helicase Dependent
Amplification HDA
12. Hot Start PCR
13. Inverse PCR
14. In Situ PCR
15. ISSR PCR
16. Single Cell PCR
17. Touchdown PCR
18. Solid Phase PCR19. Quantative PCR
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Heat causes DNA strands to separateHeat causes DNA strands to separate
3
5
5
3Denature DNA strands 94Denature DNA strands 94ooCC
5 3
3 5
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Primers bind to the template sequence
Primers bind to the template sequence
TaqTaq Polymerase recognizes doublePolymerase recognizes double--stranded substratestranded substrate
3
55
3
Primers anneal 64Primers anneal 64ooCC
3
5
5
33 535
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Extend 72Extend 72ooCC
3
53
5
35
5
3
TaqTaq Polymerase extends primerPolymerase extends primer
DNA is replicatedDNA is replicated
RepeatRepeat denaturing, annealing, and extendingdenaturing, annealing, and extending 30 cycles30 cycles
3
53 535
5
3
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The Target Product Is Made In The
Third Cycle
3
55
3
5
3
53
Cycle 2
Cycle 33
3
3
35
5
5
5
Cycle 1
3
53535
5
3
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Mycobacterium tuberculosis
Putative insertion sequence designated asIS6110
Its present in high copy numbery
Its specific and has high repetitive nature makes it idealtarget for amplification by PCR
y The internal control DNA has been constructed whichuses same primer as target sequence
y Use of internal control DNA allows assessment of theefficacy of each individual reaction ensures that thereaction is not inhibited by interfering substances
y The PCR of internal control DNA is 600bp and easilydetectable from the 123kb of the product
y DNA fingerprinting ofM tuberculosis strain has beendeveloped using probes of IS6110
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Procedure
Sample Requirementsy Sputum, bronchoalveolar lavage specimen, and
bronchial washings.
The cells are lysed DNA extracted
PCR is performed
Amplification Concentrated, separate and electrophoresis
Analysis of PCR product
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Result
Load the sample with bromophenol blue
indicator and stain it with ethidium
bromide on the 12% polyacrylamide gel
Electrophoreses at 200V
Observe under UV transilluminator
y Positive: 123 bp fragment detect
y Negative: 600 bp fragment detected
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Target
IR
3
5
IR
Bam HIPvu II
Sal I
123bp
759-778
PCR PROCUCT
75881-862
T4
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Human Immunodeficiency Virus Type I
One of the two etiologic agent
PCR is highly sensitive
Discordance in the primer pairs include
y Sequence heterogenicityy Differential analytical sensitivity of the different
primes
y Short primers capable of accommodating
mismatchesy Sampling bias
y False positive result
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Guidelines
1. Highly conserved regions to be selected andamplified
2. Primers should be relatively long
3. To accommodate mismatches should have
3terminal
4. Annealing temperature which will provide
maximum mismatch stability
5. dUTP and uracil-N-glycosylase should be
incorporated into the amplification reaction to
minimize false positive PCR. This also helps to
increase specificity in amplification
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Procedure
Collection of blood sample
Processing
RT-PCR performed
Amplification
Concentrated, separate and electrophoresis
Detection by oligomer hybridisation
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HIV-1 9.7kb
HIV-1 gag791* 2299
SK462 SK431
SK102
(1366-1395)
123bp EXPECTED PCR PROCUCT142 bp
1366 1507
SK462 SK431SK102
(1403-1435) (1507-1481)
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Cryptococcus neoformans
Its region of the ribosomal DNA (rDNA) of severalisolates ofCneoformans
Three specific primers of the fungi Cneoformans
The primers do not amplify the sequence fromother sequences
They have the potential to recognize the fungi inthe clinical specimens
PCR fingerprinting: one primer is used todistinguish between the two varieties of the fungi
This procedure allows the isolates to befingerprinted
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Type Name Nucleotice sequence , 5 TO 3 Location on target
C. Neoformans
identification
NS1 GTAGTC ATA TGC TTG TCT C 5
NA8 TTC GCA GGT TCA CCT ACGGA 3
General ITS1 TTC GTAGGT GAA CCT GCG A 5
ITS4 TCC TCC GCT TAT TGA TAT GC 3
C. Neoformans specific CN4 ATC ACC TTC CCA CTA ACA CAT T 3
CN5 GAAGGG CAT GCC TGT TTG AGAG 5
CN6 TTT AAGGCG AGC CGA CGT CCT T 3
PCR fingerprinting (GTG) Random
(GACA) Random
GAGGGT GGC GGT TCT Random
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Procedure
DNA extraction
PCR
Preperation of primersAmplification
Concentrated, separate and electrophoresis
The result is given in the figure
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Giardia Lamblia
Its is the only species being transmitted
to the succesive genration
It has a unique 18s rRNA which is one of
the most ancient
It contains many species specific
regions
Assay detects the 18s rRNA genesequence in fecal specimens obtained
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Function Name Nucleotice sequence , 5 TO 3 Location
Upstream primer JW1 GCG CAC CAGGAA TGT CTTGT 1251-1270
JW2 TCA CCT ACGGAT ACC TTG TT 1433-1414
Internal probe RDR34 AGGGAC GCG TCC GGC G 1306-1291
Primer and probe sequence for the
detection of the parasite
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Procedure
The fecal specimens collected
Sample preperation
Extraction of the DNA PCR amplification
Detection and analysis of the amplified
product
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1251-1270
JW1
1251-1270
JW2
13906-1291
Probe RDR34
Internal oligonucleotice
probe
G. Lamblia 18S rRNA gene 35
EXPECTED PCR PROCUCT183bp
1251 1433
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Advantages Disadvantages
Detect specific nucleic
acid
Sensitive, large target Independent access to
viable and nonviable
Optimises
Fingerprinting allows todiscriminate the products
Access level of gene
expression
Inhibition due to humic
substances and metals
Non specific priming Infectious and
noninfectious mixture of
the sample
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Introduction
Whiteley et al. # Wu and Wallace
Target dependent ligation
LCR employs a thermostable DNA polymeraseand thermostable DNA ligase
It uses four oligonucleotide probes of 20-24b each
Each pair of the oligonucleotide is designed tobind to the denatured target DNA
A pair of oligonucleotides are made to bind to oneof the DNA target strands
A second pair of oligonucleotides is designed tohybridize to the same regions on the
complementary DNA.
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The oligonucleotides are mixed with extractedtarget DNA, denatured
The reaction is then cooled allowing theolingnucleotide probes to bind to the target DNA
DNA polymerase fills the gap and linked by DNAligase yielding doublestranded DNA molecule
40-50bp length The cycle is repeated 30 -40 times
The power of LCR is its compatibility with otherreplication-based amplification methods.
By combining LCR with a primary amplification,one effectively lines up the crosshairs todistinguish single base-pair changes withpinpoint accuracy.
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A
T
G
A
T
C
T
A
AT
G
G
AT
GC
Denature the DNA 94C
Anneal the oligonucleotides 65C
Ligate with thermostable DNA ligase 65C
A
T
G
C
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T
A
T
A
Repeat the cycle 20-30 times
C
AT
TA
G
DNA is denatured at 94~ and the four LCRprimers anneal to their complementary strands at 65~ which is approximately 5~
below theirTin. Thermostable ligase (Q) will only ligate primers that are perfectly complementary to their target sequence and
hybridize directly adjacent to each other (as shown withL. monocytogenes, left). The discriminating bases at the 3 ends of the
upstream primers are depicted as boxes on the target as well as on the primers for clarity. Primers 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. innocua, right).
The discriminating primers have a 2-bp noncomplementary AA tail at their 5ends to avoid ligation of the 3 ends.
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Principle of LCR. (Bottom) The example shown is an LCR with matched target(L.monocytogenes) and
mismatched target(L. innocua). The pathogenic bacteria L. monocytogenes canbe distinguished from other
closely relatedListeria spp. (e.g., L. innocua) by a single base-pairdifference in the 16S rDNA/2~L.
monocytogenes has an A-T base pair at nucleotide 1258, whereas L. innocua has a G-C base pair at this
position.
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N gonorrhoeae
The assay amplification kit uses four
probes complementary to a 48-bp region
of the gonococcal opacity 1 (opa-1)
gene
(GenBankaccession name, NGOOPC
B) This gene sequence is repeated up to
11 times in the N. gonorrhoeae genome. The chosen sequence is specific for
Ngonorrhoeae.
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To resolve discrepant culture-negative,
LCR-positive specimens, another LCR assay with the pilin probe set specific
forN. gonorrhoeae was used.
The pilin probe set is specific for N. gonorrhoeae, as described previously
(1a). The specificity of the pilin LCR assay forN. gonorrhoeae was greater
than 99% when it was further evaluated with clinicalspecimens.
The pilin LCR assay procedure was similar to theN. gonorrhoeae LCR assay
procedure as described above, except that thermocyclingwas for 53 cycles of
858C for 30 s and 608C for 1 min and 100 ml of each sample diluted 1:1 with
N gonorrhoeae LCR assay transport buffer was assayed.
Specimens that gave duplicate IMx signals above 100 counts per second per
second were confirmed asN. gonorrhoeae LCR assay positive. To resolve the
results for specimens that were culture positive.
N gonorrhoeae LCR assay negative, samples were diluted with specimentransport buffer and were retested by theN. gonorrhoeae LCR assay. The
bacterial isolates, when available, were also heated in specimen transport
buffer and were tested by theN. gonorrhoeae LCR assay.
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Important factors
Design of Primers
y Single basepair overhangs
y Temperature range
y Unique primers
y Noncomplementary tails
Conditions
y One set of four primersy Thermostable ligase
y There is no extention
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Detection methods for product
y Labeled with biotin 5end and nonisotopic
reporter at 3 endy Dye include fluorescein dye in blue (FAM, 5-
carboxyfluorescein) and digoxigenin.
y anti-digoxigenin antibodies coupled to alkaline
phosphatase (AP) greatly improved thesensitivity.
y Subsequent detection of the AP could be
achieved using colorimetric, fluorescent, or
luminogenic substrates.
Applications
y Genetic , viruses and bacterial diseases
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Limitations
Background amplification due to blunt
end ligation of probe duplexes
Long reactions
Carryover contaminations
Nonstringent annealing conditions
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Types of ELISA
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
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Direct ElISA
Antigen added to solid phase, incubation
Non bound antigens washed
Antigen specific antibody with enzymeconjugate added which binds antigen
unbound antibody washed
Substrate/ chromophore added
Enzyme catalyzes reaction to give colour
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Indirect ELISA
Antigen added to solid phase, incubation
Non bound antigens washed
Antigen specific antibody added which
binds antigen
Antibodies labeled with enzyme
(conjugate) which are antispecies
Excess is washed off Substrate/ chromophore added
Enzyme catalyzes reaction to give colour
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Sandwich ELISA
Direct sandwich Indirect sandwich
Antibodies, Wash
Antigen sp, wash
Antibody enzyme
conjugaete
Substrate
Enzyme catalyzed to
give colour
Antibody, wash
Antigen sp, wash
Antibody sp to antigen
Anti-antibody enzyme
conjugate
Substrate
Enzyme catalyzed to
give colour
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Competitive ELISA Incubate antibody with antigen
Add antigen-antibody mixture to the well coated withantigen (The more antigen in the sample, the lessantibody will be able to bind to the antigen in the well,hence "competition)
Wash
Add enzyme conjugate secondary antibody
Wash
Add substrate
Enzyme catalyzes the reaction to give colour
For competitive ELISA, the higher the original antigenconcentration, the weaker the eventual signal.
The major advantage of a competitive ELISA is theability to use crude or impure samples and stillselectively bind any antigen that may be present.
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Enzymes : Chromogens /Substrates
Horse radish peroxidase
Ortho phenylene diamine/ hydrogen peroxide
2,2azino-diethylbenzaldehyde/hydrogen peroxide
Tetra methyl benzidine/ hydrogen peroxide
5-amino salicylic acid / hydrogen peroxideDiaminobenzidine/ hydrogen peroxide
Alkaline phosphatase
Para-nitro phenylphosphate
-GalactosidaseOrtho-nitrophenyl -galactosidase
Urease
Bromocresol purple
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Influenza virus H1N1
Antigen coated microtitre plate
Diluted control serum and test sample are added
If the sample contains antibodies IgG it will bindto the microtitre plate
Unbound are washed out
Enzyme conjugate: Anti human IgG peroxidaseconjugate added
Unbound antibodies washed Substrate :Tetramethylbenzidine is added
Blue coloured soluble product which turns intoyellow after adding the acid stopping solution
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M tuberculosis
ELISA methodology for measurement of
IgGAb are available.
38-Kd Ag provides serodiagnostic test
with most favorable test characteristics
described, but is limited by the lack of
purified Ag.
Serum IgGAb are observed to riseduring the first 3 months of therapy but
fall after 12-16 months.
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Reverse ELISA
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DisadvantagesAdvantages