1. DENATURATION 2. ANNEALING 3. EXTENDING

PRE FI: NUCLEIC ACID AMPLIFICATION Flashcards by Sunwoo's Berry

3 CATEGORIES OF AMPLIFICATION METHODS

TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION

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POLYMERASE CHAIN REACTION
(PCR)
- DISCOVERED IN 1983 BY?

KARY MULLIS

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WHAT CATEGORY OF NA AMPLIFICATION?

 POLYMERASE CHAIN REACTION (PCR)
- DISCOVERED IN 1983 BY KARY MULLIS
o Escherichia coli plasmid pBR322
o 1ST TO BE AMPLIFIED USER FRIENDLY, MORE
AUTOMATED AND MORE AMENABLE TO USE.
o ANY REGION OF DNA CAN BE CHOSEN

TARGET AMPLIFICATION

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COPY OF THE
TARGET DNA

AMPLICONS

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PROCEDURE OF PCR

DENATURATION
ANNEALING
EXTENDING

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DENATURATION
- SAMPLE DSDNA IS DENATURED INTO TWO SINGLE STRANDS
- HEATING AT _______ FOR
SEVERAL SECONDS TO
SEVERAL MINUTES, DEPENDING
ON THE TEMPLATE
- 100 ng to 1ug OF DNA IS USED
- FREE FROM CONTAMINATING
PROTEINS
- NO NICKS/BREAKS

94°C TO 96°C

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DNA TEMPLATE CAN BE DERIVED FROM?

PATIENT’S GENOMIC DNA
PATIENT’S MITOCHONDRIAL DNA
DNA FROM VIRUSES, BACTERIA OR PARASITES

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BASIC PROCEDURE OF PCR

2 PRIMERS WILL PRIME THE SYNTHESIS OF DNA ANNEAL (HYBRIDIZE) TO COMPLEMENTARY SEQUENCES OF THE TEMPLATE.
MOST CRUCIAL STEP
50°C to 70°C

ANNEALING

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2 primers in annealing
- hybridize in 5’

FORWARD

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2 primers in annealing
- hybridize at 3’

REVERSE

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AKA OLIGODEOXYNUCLEOTIDES
- 20-30 BASES
- DIRECTS DNA SYNTHESIS TO THE DESIRED REGION
- DETERMINE THE SPECIFICITY OF PCR
- ANALOGOUS TO PROBES
- CHEMICALLY SYNTHESIZED

PRIMERS

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MORE SPECIFIC AND HYBRIDIZE AT SLOWER RATE

LONG RPIMERS

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LESS SPECIFIC AND HYBRIDIZE AT
FASTER RATE

SHORT PRIMERS

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STARTING POINT OF ANNEALING WILL BE DETERMINED USING THE ___ OF THE PRIMER

Tm (MELTING POINT)

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FACTORS THAT MAY AFFECT THE
EXACT Tm :

REACTION CONDITIONS
SALT CONCENTRATION
TEMPLATE CONDITIONS
SECONDARY STRUCTURE ( INTERNAL FOLDING AND HYBRIDIZATION BETWEEN STRANDS)

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ABERRANT PRIMER BINDING
PRIMERS BIND TO UNINTENDED SEQUENCES
22-25C AND LEADS TO FORMATION OF MISPRIMES
PREVENTED BY HOT-START PCR

MISPRIMING

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ARTIFACT COMPOSED OF TWO PRIMERS BINDING ONTO EACH OTHER
DOUBLE THE SIZE OF THE UTILIZED PRIME

PRIMER DIMERS

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PROCEDURE OF PCR:
- DNA SYNTHESIS OCCURS
- ADDITION OF NUCLEOTIDES
(PRIMER EXTENSION)
- 68°C to 72°C (OPTIMAL TEMP OF THE ENZYME)
- POLYMERASE SYNTHESIZES A
COPY OF THE TEMPLATE DNA

EXTENSION/ELONGATION

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EXTENSION/ELONGATIION

OPTIMAL TEMP OF
THE ENZYME

68°C to 72°C

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ENZYME RESPONSIBLE FOR THE ADDITION OF NUCLEOTIDES TO THE HYBRIDIZED PRIMERS
HIGH TEMPERATURES IN DENATURATION STEP WILL INACTIVATE THE ENZYME

DNA POLYMERASE

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MAINLY UTILIZED IN PCR
FROM Thermus aquaticus
THERMOSTABLE (not easily destroyed during denaturation)
BY RANDALL SAIKI

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Taq POLYMERASE

UTILIZED IN REVERSE-TRANSCRIPTASE PCR (RNA)
FROM Thermus thermophilus

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Tth POLYMERASE

supporter
ALLOWS Taq o Tth to generate large products (30,000 bases)

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VENT POLYMERASE

PCR REAGENTS

TYPICALLY IN A PREFORMED SOLUTION CALLED?

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MASTER MATRIX (BUFFER + TEMPLATE + DNA POLYMERASE + dNTPs + KCl + MgCI + PRIMERS)

REAGENT COMPONENTS: - DIRECTS DNA SYNTHESIS TO THE DESIRED REGION

PRIMER (oligodeoxynucleotides)

REAGENTS COMPONENTS: - BUILDING BLOCKS THAT EXTEND THE PRIMERS

dATP, dGTP, dCTP, dTTP

REAGENTS COMPONENTS: - MONOVALENT CATION (SALT), FOR OPTIMAL HYBRIDIZATION OF PRIMERS TO TEMPLATE

KCL

REAGENTS COMPONENTS; - BUFFER TO MAINTAIN OPTIMAL PH FOR THE ENZYME REACTION

Tris, pH 8.4

REAGENTS COMPONENTS: - DIVALENT CATION, REQUIRED BY THE ENZYME

1.5 mM MgCl2

REAGENTS COMPONENTS: - POLYMERASE ENZYME THAT EXTENDS THE PRIMERS (ADDS DNTPS)

POLYMERASE

- A MIXTURE OF FOUR EQUIMOLAR DEOXYNUCLEOTIDE TRIPHOSPHATES  ADENINE (dATP)  THYMINE (dTTP)  GUANINE (dGTP)  CYTOSINE (dCTP) - PURITY MAY AFFECT PCR RESULTS

DEOXYNULCEOTIDE BASES

dNDP AND dMNP CAN ALSO BE A RESULT OF:

- POOR MANUFACTURING - CONTAMINATION OF HEAVY METALS

- PROVIDE OPTIMAL CONDITIONS FOR ENZYME ACTIVITY (pH 8 - 9.5) - INCREASED SALT CONCENTRATION MAY DENATURE LONG DNAS SLOWER THAN SHORTER DNAs - CONDITIONS VARY WITH PRIMERS AND TEMPLATES

PCR BUFFERS

PCR BUFFERS - MAY CHELATE Mg THAT WILL RESULT TO LOWER ENZYME EFFICIENCY

EDTA

- for optimizing reactions

ACCESSORY COMPONENTS FOR BUFFERS

ACCESSORY COMPONENTS FOR BUFFERS - LOWER DENATURING TEMPERATURE

FORMAMIDE

ACCESSORY COMPONENTS FOR BUFFERS - REDUCER - ENHANCE ENZYME ACTIVITY

DITHIOTHREITOL

ACCESSORY COMPONENTES FOR BUFFERS -BINDS INHIBITORS -ENZYME STABILIZER

BOVINE SERUM ALBUMIN

ACCESSORY COMPONENTES FOR BUFFERS - ALLOWS POLYMERASE TO ACCESS DIFFICULT AREAS

CHAOTROPIC AGENTS - TRITON X 100 - GLYCEROL - DIMETHYL SULFOXIDE

PCR EQUIPMENTS: - FIRST PCR SYSTEM CONDUCTED - REPLACED BY AUTOMATED METHODS AND THERMOSTABLE ENZYMES

WATER BATHS AND HEAT BLOCKS

PCR EQUIPMENTS: - FOR RAPID AND AUTOMATICALLY RAMP TO THE REQUIRED TEMPERATURES - EARLY VERSION:  WAX/OIL: (VAPOR BARRIERS) TO PREVENT CONDENSATION OF SAMPLE TO THE TUBE TOPS - RT PCR TYPES: - WITH FLUORESCENT DETECTORS FOR MEASUREMENT

THERMAL CYCLERS/ THERMOCYCLERS

- REAGENTS CAN BE MIXED EITHER SEPARATELY OR MASTER MIX - PRE-PCR WORK SHOULD BE PERFORMED IN A CLEAN DESIGNATED AREA - NUMBER OF CYCLES IS USUALLY 30-50 o PCR PRODUCTS CAN BE FOR: - GEL/CAPILLARY ELECTROPHORESIS - NUCLEIC ACID ANALYSIS - OTHER MOLECULAR BIOLOGY TESTS

PCR REACTIONS

CONTROLS: - ENSURES:  ENZYME IS ACTIVE  BUFFER IS OPTIMAL  PRIMERS ARE PRIMING RIGHT SEQUENCES  THERMAL CYCLERS ARE CYCLING PROPERLY

POSITIVE CONTROLS

CONTROLS: - AKA. CONTAMINATION CONTROL OR REAGENT BLANK - Negative control without DNA - Ensures that the reaction mix is not contaminated with template DNA or amplified products from previous run.

NEGATIVE CONTROL

CONTROLS: - WITH A DNA THAT HAS NO TARGET SEQUENCE THERE SHOULD BE: o NO ANNEALING - Ensures the primers are not annealing to nontarget sequences of DNA.

NEGATIVE TEMPLATE CONTROLS

CONTROLS: - CONTAINS A PRIMER BINDING SITE - DETERMINE FALSE NEGATIVES (AMPLIFICATION FAILURE)

AMPLIFICATION CONTROLS

- COMMON IN OPEN TUBE METHODS - MOST COMMON SOURCES: o PRODUCTS PREVIOUS AMPLIFICATIONS (MAY AEROSOLIZED IF UNCAPPED)

PCR CONTAMINATION

PCR CONTAMINATION CONTROL METHODS: - SEPARATE PRE AND POST PCR AREAS - POSITIVE AIRFLOW, AIRLOCKS - CHANGE OF GLOVES - UV FOR DECONTAMINATION

PHYSICAL CONTAMINATION CONTROL

ATTACH T, U, C, IN DNA UNDER UV (PREVENT DENATURATION AND AMPLIFICATION)

PSORALENS

PCR CONTAMINATION CONTROL METHODS: - FOR DECONTAMINATION OF WORKSPACES o 10% BLEACH - ENZYMATIC: DTP-UNG FOR PREVIOUS AMPLIFICATIONS - WIPE TESTS TO DETERMINE CONTAMINATION

CHEMICAL CONTAMINATION CONTROL

PRE FI: NUCLEIC ACID AMPLIFICATION Flashcards

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