Amplification Techniques In Microbiology

Nucleic Acid Amplification Techniques in Microbiology Dr.T.V.Rao MD

DNA replication Basis of Biological Evolution
DNA replication , the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA.

DNA Replication is the Basis of all Technologies
This process is "semi conservative" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule
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Understanding events in vivo Lead to vitro applications
DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA

Har Gobind Khorana, Indian Scientist, Nobel Prize Winner
Polymerase Chain Reaction (PCR), a process first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana that allows the amplification of specific DNA sequences . The improvements provided by Mullis have made PCR a central technique in biochemistry and molecular biology.

Polymerase Chain Reaction Methodology A Mile stone in Medical History
He had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times. Cetus took Mullis off his usual projects to concentrate on PCR full-time

Dr. Kary Mullis, wins Nobel Prize in 1993
Kary received a Nobel Prize in chemistry in 1993 , for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century.

How DNA Works
DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides: adenine, cytosine, guanine, and thymine.

DNA – RNA - DNA
In Molecular biology, the polymerase chain reaction ( PCR ) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence.

Create primers
To begin synthesis of a new strand, a short fragment of DNA or RNA , called a primer , must be created and paired with the template strand before DNA polymerase can synthesize new DNA.

Precise Oligonucleotide Matches the Sequences

Thermocycler is Back bone of PCR methodology
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA

Primers
Primers (short DNA fragments) containing sequences complementary to the target region along with a Primers after which the method is named) are key components to enable selective and repeated amplification.

PCR a Chain reaction
As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations .

Taq polymerase fuels the Reaction
T.aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. It replaced the DNA polymerase from E.coli originally used in PCR . Taq's optimum temperature for activity is 75-80°C, with a halflife of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C.

Bacteria Of Boiling Hot Springs In Yellowstone National Park

Boiling hot springs in Yellowstone National Park. The orange-red coloration is caused by thriving colonies of photosynthetic cyanobacteria.

Taq polymerase
Taq polymerase is a thermos table DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965. It is often abbreviated to " Taq Pol " (or simply " Taq "), and is frequently used in polymerase chain reaction (PCR), methods for greatly amplifying short segments of DNA

Drawbacks of Taq polymerase
One of Taq's drawbacks is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides. Some thermos table DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.

Disadvantages of Taq Pol
Taq Pol lacks 3’ to 5’ exonuclease proof reading activity, commonly present in
other polymerases.
Taq mis-incorporates 1 base in 10 4 .
A 400 bp target will contain an error in 33% of molecules after 20 cycles.
Error distribution will be random.

PCR - Three basic Steps
Cut
Paste
Amplify

What does PCR need?
Template (the DNA you are exploring)
Sequence-specific primers flanking the
target sequence, Forward & Reverse.
Polymerases
Nucleotides (dATP, dCTP, dGTP,
dTTP)
Magnesium chloride (enzyme cofactor)
Buffer
Water, mineral oil

Four Alphabets make what we are

DNA polymerases
DNA polymerases are a family of enzymes that carry out all forms of DNA replication. A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. .

Create primers
To begin synthesis of a new strand, a short fragment of DNA or RNA , called a primer , must be created and paired with the template strand before DNA polymerase can synthesize new DNA.

PCR Primers
TT AA C GG CC TT AA . . . TTT AAA CC GG TT
AA TT G CC GG AA TT . . . . . . . . . .>
and
<. . . . . . . . . . AAA TTT GG CC AA
TT AA C GG CC TT AA . . . TTT AAA CC GG TT

Cutting, pasting and amplifying is the basis of Reaction

DNA
Example of bonding pattern.
Primary strand
CC G AA T GGG A T G C
GG C TT A CCC T A C G
Complementary strand

What does PCR need?
Template (the DNA you are exploring)
Sequence-specific primers flanking the
target sequence, Forward & Reverse.
Polymerases
Nucleotides (dATP, dCTP, dGTP,
dTTP)
Magnesium chloride (enzyme cofactor)
Buffer
Water, mineral oil

PCR Requirements
Magnesium chloride: .5-2.5mM
Buffer: pH 8.3-8.8
dNTPs: 20-200µM
Primers: 0.1-0.5µM
DNA Polymerase: 1-2.5 units
Target DNA:  1 µg

Steps in PCR
Denaturation 93 to 95°C 1min
Annealing 50 to 55°C 45sec
Elongation 70 to 75°C 1-2min

How does PCR work?
Heat (94 o C) to denature DNA strands
Cool (54 o C) to anneal primers to template
Warm (72 o C) to activate Taq Polymerase,
which extends primers and replicates DNA
Repeat multiple cycles

Denaturation of DNA
Denaturation is the first step in PCR, in which
the DNA strands are separated by heating to
95°C.
The Hydrogen bonds between the two strands
breaks down and the two strands separates.

Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and
primers is done by cooling the DNA to 55°C.
Time taken to anneal is 45 seconds
Annealing

Taq polymerase binds ….
Taq polymerase binds to the template DNA
and starts adding nucleotides that are
complementary to the first strand.
This happens at 72°C as it is the optimum
temperature for Taq Polymerase.

Denaturing Template Heat causes DNA strands to separate Denature DNA strands 94 o C 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’

Annealing Primers
Primers bind to the template sequence
Taq Polymerase recognizes double-stranded substrate
3’ 5’ Primers anneal 64 o C 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’

Taq Polymerase Extends 3’ 5’ 3’ 5’ Extend 72 o C 3’ 5’ 3’ 5’
Taq Polymerase extends primer
DNA is replicated
Repeat denaturing, annealing, and extending 30 cycles 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’

The target product is made in the third cycle 3 ’ 5’ 3 ’ 5’ 3’ Cycle 1 Cycle 2 Cycle 3 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’

PCR Cycles Review
Denaturation: 94°- 95°C
Primer Annealing: 55°- 65°C
Elongation of DNA: 72°
Number of Cycles: 25-40
No target products are made until the third cycle.
At 30 cycles there are 1,073,741,764 target copies (~1×10 9 ).

Primers That Form Hairpins
Primers can have self-annealing regions within each primer (i.e. hairpin and fold back loops)
A primer may be self-complementary and be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
The 3´ end of the primer is base-paired,
preventing it annealing to the target DNA.

Applications of PCR differentiates the results can be compared of Individual specimens

Advantages of PCR
Speed
Ease of use
Sensitivity
Robustness

Limitations of PCR
Need for target DNA sequence information
Primer Designing for unexplored ones.
Boundary regions of DNA to be amplified must be known.
Infidelity of DNA replication.
Taq Pol – no Proof reading mech – Error 40% after 20 cycles
Short size and limiting amounts of PCR product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing projects .

How to overcome Difficulties ?
Pfu DNA Polymerase from Pyrococcus furiosus possesses 3' to 5' exonuclease proofreading activity.
The error rate is only 3.5% after 20 cycles
More amount of primer is added to avoid
primer dimering.
For unexplored genes, primers used in closely
related species are used.

REAL TIME ASSAYS

New Technologies – Real Time Assays
The Real Time assays are proving to better technologies
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization
Now a new gold standard for rapid diagnosis of virus infection in the acute phase samples.

Real-time polymerase chain reaction
Real-time polymerase chain reaction , also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction , is a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.

RT - PCR
Proving to be
Accurate
Precise
Easy to perform
RT PCR technologies are easy to transfer research Laboratory protocols to Diagnostic Laboratories

OVERVIEW of RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results

Real Time Reporters
All real time PCR systems rely upon the detection and quantization of fluorescent reporter, the signal of which increases in direct proportion of the amount of PCR product in a reaction.

REAL TIME PCR Cyber Green
USING SYBER® GREEN
The simplest and economical format the reporter is the double strand DNA specific dye SYBR ® Green
Called as Molecular Probe.

How SYBR Green works
SYBR green binds to double stranded DNA and upon excitation emits light
Thus as PCR product accumulates the fluoresce increases

Limitations of SYBER®Green
Advantages
Inexpensive
Easy to Use
Sensitive
Disadvantages
SYBR green will bind to any double stranded DNA in a reaction, may result in an overestimation of the target concentration

Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction ( RT-PCR ) is a variant of polymerase chain reaction (PCR), commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification". In RT-PCR, however, RNA strand is first reverse transcribed into its DNA complement using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR..

Not to be confused with RT- PCR
Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR), which is also sometimes (incorrectly) abbreviated as RT-PCR.

Other Emerging Alternatives
Two most popular alternatives to SYBR green are TaqMan® and Molecular Beacons .
Both technologies depend on hybridization probes relying on fluorescence resonance energy transfer.( FRET) and quantization

TaqMAN ®

TaqMAN ® Sequencing

TaqMAN ® probes

Documentation of Amplification
The light emitted from the dye in the excited state is received by a computer and shown on a graph display, such as this, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis.

Molecular Beacons
Molecular Beacons
Uses FRET Fluorescence Resonance Energy Transfer
Uses two sequence specific
Oligonucleotide labelled with fluorescent dyes

Molecular Beacons – RT PCR
Molecular beacons are designed to adopt a hairpin structure while free in solution, brining the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target the fluorescent dye emits light upon irradiation, and rebind to target in every cycle for signal measurement.

Loop Mediated Isothermal Amplification (LAMP)
Loop mediated isothermal amplification is a simple, rapid, specific and cost effective nucleic acid amplification method characterized by use of 8 distinct regions on the target gene.
The amplification proceeds at a constant temperature using strand displacement reaction.

LAMP
Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers DNA polymerase with strand displacement activity and substrates at a constant temperature of 63 0 c.

LAMP
Compared with PCR, and real time PCR, the LAMP has advantages of reaction simplicity and detection sensitivity.
The higher sensitivity and specificity of LAMP reaction is attributed to continuous amplification under isothermal condition employing six primers recognizing eight distinct regions of the target.

Advantages of LAMP
LAMP functions on isothermal amplification.
LAMP does not require any thermal cycler and thus cane be performed even with water bath/heating block
LAMP method do not require sophisticated temperature control devices
Cost effective

Lesser False Positives in LAMP
In LAMP both amplification and detection occur simultaneously during the exponential phase without going through the plateau phase where the non spurious amplification leads to lower sensitivity and false positivity.

Loop Mediated Isothermal Amplification in Clinical Diagnosis
LAMP technology proving to be ideal in detection of DNA or RNA of the pathogenic organisms
Proving to be highly efficient in diagnosis of Viral and Bacterial infections,
LAMP is capable of detecting the presence of pathogenic agents earlier than PCR

LAMP proving an efficient Technology
A one step single tube real time accelerated reverse transcription loop mediated isothermal amplification (RT-LAMP ) assays for rapid detection of some recently emerged viral pathogen eg West Nile, SARS, Dengue, Japanese encephalitis Chikungunya Norwalk, H5N1 highly pathogenic avian influenza., and CMV,HPV,VZV

Multiplex PCR
TaqMan probes and Molecular beacons allow multiple DNA species to be measured in the same sample ( Multiplex PCR) since fluorescent dyes with different emission spectra may be attached to different probes

Uses of Automated RT - PCR
Several viral infections can be detected in acute phase serum samples.
Increasing used in for early and accurate detection of all most human viruses including
Measles, Mumps, Herpes simplex viruses, Rota viruses Noro virus,Influenzae virus type A and B, Respiratory Syncitical virus, SARS, Dengue Japanese Encephalitis, Hepatitis B and C, West Nile, Chikungunya,HIV, Avian flu virus,

Multiplex PCR in Real Time
Multiplex real time quantitative RT-PCR assays have been developed for simultaneous detection identification and quantification of HBV, HCV and HIV-! In plasma and Serum samples.

Real-Time PCR Method Molecular Beacons
Molecular beacons are short segments of single-stranded DNA . The sequence of each molecular beacon must be customized to detect the PCR product of interest.

Molecular methods are necessary if the traditional methods provide poor results
Microscopy gives false positive results -
- T.vaginalis, N.gonorrhoeae
Intracellular pathogens – viruses, M.genitalium
Low sensitivity – Chlamydia sp.,Neisseria sp.
Seropositivity is common – Chlamydia sp .
Subtyping is mandatory – HSV, HPV, HCV
Microbial growth is slow – M. tuberculosis

How are you going to measure the PCR product
Directly
Sybr green
Quality of primers critical
Indirectly
In addition to primers, add a fluorescently labeled hybridization probe

Importance of controls
Negative control (no DNA)
checks reagents for contamination
No reverse transcriptase control
detects if signal from contaminating DNA
Positive control
checks that reagents and primers work
especially importance if trying to show absence of expression of a gene

Standards
Same copy number in all cells
Expressed in all cells
Medium copy number advantageous
correction more accurate
Reasonably large introns
No pseudo gene
No alternate splicing in region you want to PCR

Real-time PCR applications
Quantitation of gene expression
Pathogen detection
Viral quantitation
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Genotyping

Emerging Technologies in Molecular Diagnosis

QIAGEN One Step RT-PCR Kit
The QIAGEN One Step RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template. A unique enzyme combination and specially developed reaction buffer ensure efficient reverse transcription and PCR in one tube.

RT-PCR in one step The Robus™ T I Kit is base
RobusT RT-PCR Kits perform cDNA synthesis and PCR amplification of cDNA successively in a single tube during a continuous thermal cycling

Uses and Advantages in Testing by PCR Methods
Clinical diagnostics: detection and quantification of infectious microorganisms, cancer cells and genetic disorders
Capable of amplifying long targets, up to 6.0 kb
One-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination
Amplifies products from a wide variety of total RNA or mRNA sources

Advantages Molecular methods
High sensitivity and specificity
Detects pathogen, not immune response
Quick results
High transport toleration
In-house (home-brew) PCR methods
Cost effective
High sensitivity
High quality
Fast implementation of scientific discoveries
Customer friendly
R&D is absolutely necessary

ESTABLISHMENT OF A PCR LABORATORY
To perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required. The specific technical operations, reagents ,and personnel considerations

Prevention of Contamination in PCR Laboratory
PCR contamination be considered as a form of infection . If standard sterile techniques that would be applied to tissue culture or microbiological manipulations are applied to PCR, then the risk of contamination will be greatly reduced. Above all else, common sense should prevail.

Avoiding contamination
The single most important source of PCR product contamination is the generation of aerosols of PCR amplicons that is associated with the post-PCR analysis. Methods for eliminating this aerosol range from physical design of laboratories and use of specific pipettes to chemical and enzymatic approaches. The choice of method is often dependent on the frequency of amplification of a target amplicon and the relative amounts and concentrations of the amplicons created by the PCR.

PCR laboratory Sample handling DNA preparation Clean room Stock solutions Laboratory Mixing site Thermocycler Amplification Detection Documentation QC & QA Quality control & assurance R & D ( Research and development) Alternatives: - commercial kits - robots + kits No alternative

Aseptic Handling of Specimen a Top Priority

Machines can be Operated in limited Space

Contamination
PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules of DNA. The sensitivity of PCR means that the sample used for PCR should not be contaminated with any other DNA’s that may reside in the laboratory environment.

C an we do better ?
Create networking between laboratories
Take second opinion
Honest dialogue with doctors
Education
Concentration into specialized laboratories
QC + QC + QC – to build up the trust

A Drop of Blood decides Human Destiny

Conclusion
PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA in infectious diseases, but it is also important for genetic predisposing for defects in Genetic disorders.
The PCR technology can also be employed in law enforcement, genetic testing of animal stocks and vegetable hybrids, and drug screening along with many more areas.

Dr. Kary Banks Mullis on Wisdom of Creative Nature
Science, like nothing else among the institutions of mankind, grows like a weed every year.
We not only can luxuriate in its weed-like growth, but we can each of us be a creative and active part of it if we so desire.
There is no stopping it, nor can there be any end to it."

Playing well with Genes Can Change Life ?

Created for Awareness in Developing World on Emerging Technologies in Microbiology Dr.T.V.Rao MD Email [email_address]

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Amplification Techniques In Microbiology

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