PRE FI: NUCLEIC ACID AMPLIFICATION Flashcards
3 CATEGORIES OF AMPLIFICATION METHODS
TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION
POLYMERASE CHAIN REACTION
(PCR)
- DISCOVERED IN 1983 BY?
KARY MULLIS
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
COPY OF THE
TARGET DNA
AMPLICONS
PROCEDURE OF PCR
- DENATURATION
- ANNEALING
- EXTENDING
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
DNA TEMPLATE CAN BE DERIVED FROM?
- PATIENT’S GENOMIC DNA
- PATIENT’S MITOCHONDRIAL DNA
- DNA FROM VIRUSES, BACTERIA OR PARASITES
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
2 primers in annealing
- hybridize in 5’
FORWARD
2 primers in annealing
- hybridize at 3’
REVERSE
AKA OLIGODEOXYNUCLEOTIDES
- 20-30 BASES
- DIRECTS DNA SYNTHESIS TO THE DESIRED REGION
- DETERMINE THE SPECIFICITY OF PCR
- ANALOGOUS TO PROBES
- CHEMICALLY SYNTHESIZED
PRIMERS
MORE SPECIFIC AND HYBRIDIZE AT SLOWER RATE
LONG RPIMERS
LESS SPECIFIC AND HYBRIDIZE AT
FASTER RATE
SHORT PRIMERS
STARTING POINT OF ANNEALING WILL BE DETERMINED USING THE ___ OF THE PRIMER
Tm (MELTING POINT)
FACTORS THAT MAY AFFECT THE
EXACT Tm :
- REACTION CONDITIONS
- SALT CONCENTRATION
- TEMPLATE CONDITIONS
- SECONDARY STRUCTURE ( INTERNAL FOLDING AND HYBRIDIZATION BETWEEN STRANDS)
- ABERRANT PRIMER BINDING
- PRIMERS BIND TO UNINTENDED SEQUENCES
- 22-25C AND LEADS TO FORMATION OF MISPRIMES
- PREVENTED BY HOT-START PCR
MISPRIMING
- ARTIFACT COMPOSED OF TWO PRIMERS BINDING ONTO EACH OTHER
- DOUBLE THE SIZE OF THE UTILIZED PRIME
PRIMER DIMERS
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
EXTENSION/ELONGATIION
- OPTIMAL TEMP OF
THE ENZYME
68°C to 72°C
- ENZYME RESPONSIBLE FOR THE ADDITION OF NUCLEOTIDES TO THE HYBRIDIZED PRIMERS
- HIGH TEMPERATURES IN DENATURATION STEP WILL INACTIVATE THE ENZYME
DNA POLYMERASE
- MAINLY UTILIZED IN PCR
- FROM Thermus aquaticus
- THERMOSTABLE (not easily destroyed during denaturation)
- BY RANDALL SAIKI
Taq POLYMERASE
- UTILIZED IN REVERSE-TRANSCRIPTASE PCR (RNA)
- FROM Thermus thermophilus
Tth POLYMERASE
- supporter
- ALLOWS Taq o Tth to generate large products (30,000 bases)
VENT POLYMERASE
PCR REAGENTS
- TYPICALLY IN A PREFORMED SOLUTION CALLED?
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
