PRE FI: PCR MODIFICATION ANALYSIS Flashcards
ADAPTED FOR VARIOUS
APPLICATIONS
MODIFIED PCR
TYPES OF MODIFIED PCR
- MULTIPLEX PCR
- SEQUENCE SPECIFIC PCR
- REVERSE TRANSCRIPTASE PCR
- NESTED PCR
- REAL - TIME PCR
- OTHER TECHNIQUES (TOUCHDOWN PCR & HOT START PCR)
- PRIMERS CAN BE “MULTIPLE” (MORE THAN A PAIR)
- APPLICATION:
o MULTIPLE AMPLIFIED
TARGETS FOR HUMAN/
PATHOGEN/FORENSIC:
TYPING
IDENTIFICATION ANALYSES
MULTIPLEX PCR
DISADVANTAGE OF MULTIPLEX PCR
PRIMERS MAY EXHIBIT SELF-INHIBITION (INTERFERE WITH EACH OTHER)
- IDENTIFIES THE “SINGLE-BASE CHANGE” IN THE TARGET DNA
- EITHER FORWARD OR REVERSE PRIMERS ARE DESIGNED WITH A 3’ BASE COMPLEMENTARY TO MUTANT
SEQUENCE - APPLICATION:
o HUMAN LEUKOCYTE ANTIGEN (HLA) ALLELE ANALYSIS IN TISSUE TYPING
SEQUENCE SPECIFIC PCR
STARTING MATERIAL OF REVERSE TRANSCRIPTASE PCR
RNA
o FROM RNA VIRUSES
o :COPIES RNA INTO HYBRID RNA”: DNA THEN USES HAIRPIN FORMATION TO DNA STAND → CREATE STRAND TO REPLACE THE
RNA→ cDNA (A DSDNA) WILL BE AMPLIFIED
REVERSE TRANSCRIPTASE PCR
REVERSE TRANSCRIPTASE PRIMERS CAN BIND TO:
- mRNA’s RANDOM SEQUENCE
- HEXAMERES/DECAMERES : 6 OR 10
BASE LONG PRIMERS - mRNA WITH POLY A TAILS
o 18-BASE LONG PRIMERS REVERSE
TYPES of RT-PCR:
- USES Tth POLYMERASE
- RNA SAMPLE IS DIRECTLY APPLIED
- INCLUDES INITIAL INCUBATION FOT RT (45- 50C FOR 30-60MINS)
ONE - STEP
TYPES of RT-PCR:
- CDNA SYNTHESIS → PCR
TWO - STEP
APPLICATION OF RT PCR
GOLD STANDARD for detection of SARS - COV 2/COVID 19
DISADVANTAGES of RT-PCR
RNA IS VULNERABLE TO DEGRADATION (ENVIRONMENTAL RNASES)
- INCREASES SENSITIVITY AND SPECIFICITY OF THE REACTION
- USES TWO PAIRS OF PRIMERS FOR A SINGLE TARGET
- SECOND PAIR WILL BIND SLIGHTLY INSIDE THE BINDING SITES OF FIRST PAIR
NESTED PCR
TYPE OF NESTED PCR:
- ONE OF EXTERNAL PRIMERS IS USED ON ‘SECOND ROUND’
SEMI - NESTED PCR
TYPE OF NESTED PCR:
- DIFFERENT PAIR OF PRIMERS ARE USED
TRADITIONAL NESTED PCR
DISADVANTAGE OF NESTED PCR
- INCREASED OF CONTAMINATION
- LABOR - INTENSIVE
- TIME CONSUMING
- AKA QUANTITATIVE PCR (qPCR)
- TO DETERMINE THE AMOUNT OF TARGET SEQUENCE PRESENT
- PRINCIPLE: UTILIZES DYES IN DETECTING FLUORESCENCE REACTION OF PCR PRODUCTS AS THEY ARE MADE AND MONITORED REAL-TIME
REAL - TIME PCR
REAL - TIME PCR is plotted in?
SIGMOIDAL CURVE
o X AXIS - PCR CYCLES
o Y AXIS- FLUORESCENCE GENERATED
REAL - TIME PCR AMPLIFICATION CONTROL:
- cycles 0 - 15
- start of the run, amount of PCR product is LOW → very little fluorescence
BACKGROUND/BASELINE
REAL - TIME PCR AMPLIFICATION CONTROL:
- cycles 15 -25
- amount of PCR product DOUBLES for every PCR cycle
EXPONENTIAL PHASE
REAL - TIME PCR AMPLIFICATION CONTROL:
- cycles 30 - 40
- once all reagents have been used up, AMPLIFICATION WILL SLOW, no more PCR procedures → fluorescence signal stabilizes
PLATEAU
- cycle number when the fluorescence of a PCR product can be detected above the background signal
- value is INVERSELY PROPORTIONAL to the amount of target NA (present in the sample)
- can be used to quantify the amount of target DNA in the sample
- APPLIES TO qPCR, AND RT-qPCR
THRESHOLD CYCLE (CT)
REAL - TIME PCR
FLUORESCENT AND DYE-BINDING DYES ARE USED:
o BINDS TO NONSPECIFIC REGIONS OF DNA
o MORE COPIES, MORE FLUORESCENCE
o REQUIRES TO PREVENT MISPRIMING TO PREVENT FLUORESCENCE OF THOSE
NONSPECIFIC
REAL - TIME PCR
FLUORESCENT AND DYE-BINDING DYES ARE USED:
o HYBRIDIZE TO SEQUENCES BETWEEN PRIMER AND DNA TEMPLATE
o EXPLOITS THE Taq POLYMERASE’S
EXONUCLEASE ACTIVITY (5’-3’) GENERATED BY SEPARATION OF 2 UNITS:
- QUENCHER
- REPORTER
TaqMan PROBE
INHIBITS FLUORESCENCE WHEN BOUND TO REPORTER
QUENCHER (3’ END)
‘YIELDS FLUORESCENCE’ WHEN QUENCHER IS SEPARATED
REPORTER (5’ END)
REAL - TIME PCR
- MEASURES ACCUMULATION OF PRODUCT AT ANNEALING STEP IN THE PCR CYCLE
- 25 BASES LONG HAIRPIN/STEM AND LOOP APPEARANCE
- HYBRIDIZATION PROMOTES OPENING AND ACTIVATION
MOLECULAR BEACONS
0 FOR FAST CYCLING CONDITIONS
- USEFUL IN FURTHER ANALYSIS LIKE CAPILLARY ELECTROPHORESIS
SCORPION-TYPE PRIMERS
- USES 2 SPECIFIC PROBES THAT BIND TO ADJACENT TARGETS
FRET (FLUORESCENT RESONANCE ENERGY TRANSFER)
(ACCEPTOR/REPORTER FLUOR)
3’ FLUOROPHORE
(DONOR FLUOR)
5’ CATALYST
DISADVANTAGES REAL-TIME PCR:
o CANNOT DIFFERENTIATE LIVE OR DEAD CELLS
o PRIMER DIMERS MAY INTERFERE WITH DETECTION (FALSE INCREASED)
o COST OF PRIMERS
CONVENTIONAL PCR
DATA ANALYSIS:
QUANTIFICATION::
TURNOVER TIME:
- END POINT
- ELECTROPHORESIS
- LONGER ( PCR + ELECTROPHORESIS)
REAL - TIME PCR
DATA ANALYSIS:
QUANTIFICATION::
TURNOVER TIME:
- EXPONENTIAL PHASE OF GROWTH
- DYES
- SHORTER (SIMULTANEOUS MEASUREMENT AND AMPLIFICATION
- ENHANCEMENT OF PCR PRODUCTS
- ANNEALING TEMPERATURES HIGHER THAN USUAL AND DECREASED 1C EVERY CYCLE/EVERY OTHER CYCLE UNTIL OPTIMAL ANNEALING TEMPERATURE IS REACHED
- MODIFICATIONS:
TOUCHDOWN PCR
LOWERS 5C INSTEAD OF 1C
STEPDOWN PCR
ADJUSTMENT OF RAMP SPEED AND COOLING RATE OF THERMAL
CYCLERS
SLOWDOWN PCR
APPLICATION OF TOUCHDOWN PCR
- GENE CLONING
- SITE - DIRECTED MUTAGENESIS
- DETECTION OF NON - ABUNDANCE TARGETS
- METHOD FOR PREVENTION OF MISPRIMING
- REDUCES PREMATURE DNA POLYMERASE ACTIVITY DURING THE INITIAL STAGES OF THE REACTION SET - UP
HOT START PCR
APPLICATIONS OF HOT START PCR
- GENE CLONING
- MUTATIONAL ANALYSIS
- TEMPLATE GENERATION FOR SEQUENCING
- DETECTION OF LOW-COPY NUMBER OF TARGETS
