- The first prototypical (specific) method for amplifying target nucleic acids. - Have been developed to amplify nucleic acid can be divided into groups based on:

- POLYMERASE CHAIN REACTION (PCR) - Whether the target nucleic acid itself - Probe specific for the target sequence - Signal used to detect target nucleic acid is amplified

NUCLEIC ACID AMPLIFICATION TECHNIQUES (PART 1) Flashcards by Queenie Mae

Nucleic acid (NA) amplification methods fall into three categories:

Target Amplification – targets DNA and RNA
Probe Amplification
Signal Amplification

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synonymous to primer

Probe

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binds to ssDNA/RNA
Usually _ nucleotides
May or may not have probes

primer
- 10

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segment of DNA that will hybridize to a specific DNA
Bind to __
Usually 10-1000 nucleotides
Has fluorophore
Used to pinpoint a specific segment of DNA

probe
- dsDNA

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Involves making copies of a target sequence to such a level (in millions of copies) that they can be detected in vitro.
This is analogous to growing cells in the culture and allowing the cells to replicate nucleic acids, as well as themselves.

TARGET AMPLIFICATION

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Nowadays, the gold standard for diagnosing infectious diseases is _____________

molecular techniques

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is only a matter of hours compared to culturing.

molecular studies

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is at the top of biological test

Molecular test

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Target Amplification Sequence

• 1st: Molecular assay - 2nd: Biological assay - 3rd: Serological assay

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The first prototypical (specific) method for amplifying target nucleic acids.
Have been developed to amplify nucleic acid can be divided into groups based on:

POLYMERASE CHAIN REACTION (PCR)
Whether the target nucleic acid itself
Probe specific for the target sequence
Signal used to detect target nucleic acid is amplified

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It has become increasingly - user friendly, more automated, amenable to use in a clinical laboratory setup with infinite applications possible.

POLYMERASE CHAIN REACTION (PCR)

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Nowadays, PCR used for ___________
- gold standard

SARS-CoV detections

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Usually __________ for PCR SARS-COV 2

45 cycles

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conceived the idea of amplifying DNA in vitro.

KARY MULLIS
1983

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In the process of working through a mutation detection method, He came up on a way to double the test target.

Mullis

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Knew that a cheap, commercial enzyme was available (short fragment of __).

E. coli DNA polymerase

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He called the method ____________________ because the DNA polymerase was the enzyme, he used to drive the replication of the DNA.

Karry Mullis
- Polymerase Catalyze Chain Reaction

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Replication continued in a chain reaction, then name is shortened to PCR: before called__

THEN: Polymerase Catalyze Chain Reaction

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Three Graduated Student

Denaturing – 94-96°C (20-60 secs)
Annealing – 50-70°C (20-90 secs)
Extending – 68-75°C (10-60 secs)

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Knew that you could expose template DNA by ______________ to produce single-stranded DNA.

boiling double-stranded DNA

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Restriction Digest

RFLP (Restriction Fragment Length Polymorphism)

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circular strand of DNA

Plasmid

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❑__ - The first successful amplification.

E. coli plasmid (PBR322)

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In cases of false positive reactions, remove ___________ and replace it with __

thymine
uracil

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* One copy of dsDNA becoming two copies. * About an hour or two, PCR can produce millions of copies called amplicons of DNA.

In vitro

DNTPS/NITROGENOUS BASES:

Adenine, Thymine, Guanine, Cytosine

catalyze addition of uracil

Uracil N-glycosylase (UNG)

it would probably take several days for a cell to produce the same number of copies_

in vivo

Reduce double stranded molecules to single stranded molecules.

Denaturation

Denaturation is accomplished by heating the samples by _ for __ seconds up to several minutes, depending on the type of target for testing.

94-96°C for 20-60 seconds

lengthened for genome-make or other large template fragments.

Initial Denaturation Step

DNA pol adds subsequent bases.

TAQ POLYMERASE

can be shorter depending on the type of template being used.

Subsequent Denaturation

Temperatures used in PCR:

- 95°C - 55°C - 72°C

Controls specificity of hybridization. MOST CRITICAL STEP for the specificity of the PCR.

Annealing

dictates the part of the template that would be amplified. determines the specificity of the amplification.

Primer

❑ This is when the DNA synthesis occurs. ❑ In this step, the polymerases synthesize a copy of the template DNA by adding a nucleotide to the hybridized primers. ❑ The DNA Polymerase catalyzes the formation of the phosphodiester bonds in between the incoming DNTPs which is then determined by the hydrogen bonding to the template. ❑ Either AT or GC ❑ Base at the 3’ end of the primer.

EXTENSION (PRIMER EXTENSION)

* In some cases, the annealing temperature is close enough to the extension temperature that the reaction can proceed with only 2 temperature changes.

- TWO-STEP PCR

COMPONENTS OF PCR

• Primers • DNA Template • DN Bases • DNA Pol. • PCR Buffer • Thermal cyclers • Controls for PCR

❑ Critical component of PCR because they determine the specificity of the PCR. ❑ They are analogous to the probes in blotting and hybridization procedures. ❑ Designed to contain sequences homologous to sites flanking the region to be analyzed. ❑ Single-stranded DNA fragments

PRIMERS

* May be single or double stranded DNA * Clinical sample sources - human (genomic samples), bacteria, virus * __- only one or two copies PCV of single copy genes - serve as amplification targets. * PCR reagents and conditions, _ amounts of genomic DNA are - sufficient for consistent results.

- DNA TEMPLATE - Genomic DNA nanogram

* Routine Clinical Analysis – ___________ of DNA is usually used. - Lesser amounts are required for more defined template preparations such as ___________ or product from a previous amplification.

- 100 ng to 1 μg - cloned target DNA

(Guanine-Cytosine) has ____________

- 3 nitrogen bonds

are the building blocks of DNA.

Nucleotide triphosphates

❑ Deoxyribonucleotide triphosphates (dNTPs)

1. Adenine 2. Thymine 3. Guanine 4. Cytosine

* Were subsequently exploited for laboratory use. * Also has reverse transcriptase activity  can be used in reverse transcriptase PCR or RT-PCR. * Can be found in viruses * Function: Converts RNA to cDNA (complimentary DNA)

TTH POLYMERASE * Thermus thermophilus

In common PCR applications, the recommended final concentration of each dNTP is generally__

0.2 mM

* Provides optimal conditions for enzyme activity. * The buffer pH is usually between 8.0 and 9.5 and is often stabilized by _

PCR BUFFER Tris-HCl

▪ Potassium chloride (20-100 mM) ▪ Ammonium sulfate (15-30 mM) ▪ hybridize primer to template

Monovalent Cation

▪ Magnesium chloride (conc: 1-4 mM)  affects primer annealing and is very important for enzyme activity. ▪ increases activity of Taq

Divalent Cation

• Most commonly used buffer • Stabilized using HCl • Add chelating EDTA (chelates Mg & Ca ions)

Tris

– isolated from Thermus aquaticus ❑Using an enzyme derived from a thermophilic bacterium meant that the DNA polymerase could be added once at the beginning of the procedure and it would maintain its activity throughout the heating and cooling cycles.

Taq Polymerase

Recommended concentrations:

▪ Mg = 1-4 mM ▪ Buffer for Tris = 10 mM

❑Have proof-reading functions and can generate products over 30kbp (30,000 bases in length)

Vent Polymerase – Thermococcus litoralis

* ____________ - lower enzyme efficiency, resulting in a low yield of PCR product.

- Too few Mg2+ ions

promote misincorporation and thus increase the yield of nonspecific products.

Overly high Mg2+ concentrations:

: are desirable when fidelity of the PCR is critical.

Lower Mg2+ concentrations

ACCESSORY COMPONENTS – USED TO OPTIMIZE REACTIONS

- BOVINE SERUM ALBUMIN - DITHIOTHREITOL - FORMALDEHYDE

o 10-100 ug/ml o Binds inhibitor o Stabilizes enzyme

BOVINE SERUM ALBUMIN

o 0.1 mM o reducing conditions that may enhance enzyme activity

DITHIOTHREITOL

o 1-10% o lower denaturing temp o high secondary structure o increase availability for primer binding

FORMALDEHYDE

First PCRs were performed using multiple water baths or heat blocks set at the required temperatures for each of the steps.

THERMAL CYCLERS

Running the correct controls in PCR is essential for maintaining and ensuring the accuracy of the assay. ❑ With every PCR run, the appropriate controls must be included.

- CONTROLS OF PCR

* Ensure that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately

POSITIVE CONTROL

ensures that the reaction mix is not contaminated with template DNA or amplified product from a previous run.

❑NEGATIVE CONTROLS (CONTAMINATION CONTROL/REAGENT BLANK)

❑Widely used method for decontamination and preparation of the workspace. ❑ As a common practice in forensic work, before handling items that come in contact with evidence, gloves are wiped with bleach and is allowed to air-dry.

❑ 10% Bleach solution (7mM sodium hypochlorite)

▪ __________ - catalyzes production of s protein ▪ __________ - catalyzes production of envelope CHON ▪ __________ - catalyzes production nucleocapsid CHON

- Genes - S gene - Envelope gene - Nucleocapsid gene

What if positive becomes negative?

• Contaminated

PCR MODIFICATIONS

- Multiplex PCR - Reverse Transcripticase PCR - Nested PCR - Real-Time (Quanti) PCR - Arbitraty Primed PCR - Transcription-Based Ampli. System

• PCR today has been adapted for various applications. • Several modifications are used in the modern clinical Laboratories.

PCR MODIFICATIONS

❑Can be used in detecting MRSA (methicillin resistant Staphylococcus aureus)

MULTIPLEX PCR

❑ Amplification by PCR requires a double-stranded DNA template ❑ Used if the starting material for procedure is RNA ❑If the starting material for a procedure is RNA, it must first be converted to double-stranded DNA.

REVERSE TRANSCRIPTASE PCR (RT-PCR)

an enzyme isolated from RNA viruse

❑ Reverse transcriptase – PCR

This enzyme first copies the RNA single strand into a RNA:DNA hybrid strand and then uses a hairpin the hybrid. formation on the end of the newly synthesized DNA strand to prime synthesis of the homologous DNA strand, replacing the original RNA in 2. The resulting double-stranded DNA is called __

REVERSE TRANSCRIPTASE PCR (RT-PCR) - cDNA (copy or complementary DNA)

RT-PCR was originally performed in two steps:

1. cDNA synthesis 2. PCR

, which has RT activity and proprietary mixtures of RT and sequestered (hot-start) DNA polymerase, are components of one step RT-PCR procedures. ❑These methods are more convenient than the two-step procedure, as RNA is added directly to the PCR. ❑The amplification program is modified to include an initial incubation of 45 50C for 30-60 minutes during which RT makes cDNA from RNA in the sample. ❑The RT activity will then be inactivated in the first denaturation step of the PCR procedure.

Tth DNA polymerase

more than one primer pair can be added to a PCR so that multiple amplifications are primed simultaneously, resulting in the formation of multiple products. Multiple organisms have been the target

MULTIPLEX PCR

the two pairs of primers are used to amplify a single target in two separate PCR runs. * The second pair of primers, designed to bind slightly inside of the binding sites of the first pair of primers, will amplify the product of the first PCR in a second round of amplification. * The second amplification will specifically increase the amount of the intended product.

NESTED PCR

, one of the second-round primers is the same as the first-round primer.

semi-nested PCR

* Performed by adding ethidium bromide (EtBr) to a regular PC

REAL-TIME (QUANTITATIVE) PCR

- Can be used to monitor accumulation of PCR products during the reaction in real time - This dye intercalates into dsDNA and fluoresce * Advantage - determine amount of starting template accurately

* Ethidium bromide (EtBr)

1. The _ procedure measures the fluorescent signal generated by separation of fluorescent dye and quencher - single-strandedwhich is covalently attached to a fluorescent dye on the 5 ′ end and another dye or nonfluorescent molecule that pulls fluorescent energy from the 5 ′ dye (quencher) on the 3 ′ end

Probe based systems of RTPCR - TaqMan

measures the accumulation of product at the annealing step in the PCR cycle

molecular Beacons

utilizes two specific probes, one with a 3 ′ fluorophore (acceptor) and the other with a 5 ′ catalyst for the fluorescence (donor), that bind to adjacent targets

fluorescent resonance energy transfer (FRET),

Examples of frequently used donor acceptor pairs are _,_ and _

fluorescein rhodamine, fluorescein–(2 aminopurine), and fluorescein–Cy5

Commercial Variations:

1. Transcription-Mediated Amplification (TMA) 2. Nucleic Acid Sequence-Based Amplification (NASBA) 3 3. Self-sustaining Sequence Replication (3SR)

❑Amplification involves synthesis of cDNA from RNA target with a primer containing the_

T7 RNA pol promoter sequence

- Procedure with slight modification on an RNA Target -uses three enzymes:

- Nucleic Acid Sequence-Based Amplification (NASBA) - Reverse Transcriptase - RNase H - T7 RNA polymerase)

Deoxyribonucleotide triphosphates (dNTPs):

- Adenine – deoxyadenosine TP - Thymine – deoxythymidine TP - Guanine – deoxyguanosine TP - Cytosine – deoxycytidine TP

❑Useful in typing or identification analyses. ❑ Individual organisms, from viruses to humans, can be identified or typed by observing a set of several PCR products at once.

MULTIPLEX PCR

AKA Randomly Amplified Polymorphic DNA (RAPD) or Random Amplification of Polymorphic DNA ❑ PCR products are generated without the sequence of the target gene ❑Possible to obtain multiple products that depends on how many times a short sequence appear on the Genome ❑Used primarily in the epidemiological typing in the microorganisms

Arbitrary Primed PCR

is designed to simultaneously amplify thousands of specific templates in a single reaction, producing a set of specific products, or library. In this method, the ends of fragmented template DNA are ligated to universal primer sequences and, along with the other components of the PCR reaction (buffer, polymerase enzyme, forward and reverse primers, dNTPs), are added to an oil surfactant mixture (Span 80, Tween 80, Triton X-100, and mineral oil

Emulsion PCR

Detection of M. tuberculosis (Respiratory samples) ❑In smear-positive respiratory samples ❑ Detection of C. trachomatis (Genital Specimens) ❑ Detection of HIV and CMV (Blood) ❑ Ribosomal typing ❑ Detection of microbial pathogens in food and environmental sample

❑Nucleic Acid Sequence-Based Amplification (NASBA)