PRE FI: PCR MODIFICATION ANALYSIS

Question 1

ADAPTED FOR VARIOUS
APPLICATIONS

Answer 1

MODIFIED PCR

Question 2

TYPES OF MODIFIED PCR

Answer 2

• MULTIPLEX PCR
• SEQUENCE SPECIFIC PCR
• REVERSE TRANSCRIPTASE PCR
• NESTED PCR
• REAL - TIME PCR
• OTHER TECHNIQUES (TOUCHDOWN PCR & HOT START PCR)

Question 3

• PRIMERS CAN BE “MULTIPLE” (MORE THAN A PAIR)
• APPLICATION:
  o MULTIPLE AMPLIFIED
  TARGETS FOR HUMAN/
  PATHOGEN/FORENSIC:
   TYPING
   IDENTIFICATION ANALYSES

Answer 3

MULTIPLEX PCR

Question 4

DISADVANTAGE OF MULTIPLEX PCR

Answer 4

PRIMERS MAY EXHIBIT SELF-INHIBITION (INTERFERE WITH EACH OTHER)

Question 5

• 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

Answer 5

SEQUENCE SPECIFIC PCR

Question 6

STARTING MATERIAL OF REVERSE TRANSCRIPTASE PCR

Answer 6

RNA

Question 7

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

Answer 7

REVERSE TRANSCRIPTASE PCR

Question 8

REVERSE TRANSCRIPTASE PRIMERS CAN BIND TO:

Answer 8

• mRNA’s RANDOM SEQUENCE
• HEXAMERES/DECAMERES : 6 OR 10
  BASE LONG PRIMERS
• mRNA WITH POLY A TAILS
  o 18-BASE LONG PRIMERS REVERSE

Question 9

TYPES of RT-PCR:
- USES Tth POLYMERASE
- RNA SAMPLE IS DIRECTLY APPLIED
- INCLUDES INITIAL INCUBATION FOT RT (45- 50C FOR 30-60MINS)

Answer 9

ONE - STEP

Question 10

TYPES of RT-PCR:
- CDNA SYNTHESIS → PCR

Answer 10

TWO - STEP

Question 11

APPLICATION OF RT PCR

Answer 11

GOLD STANDARD for detection of SARS - COV 2/COVID 19

Question 12

DISADVANTAGES of RT-PCR

Answer 12

RNA IS VULNERABLE TO DEGRADATION (ENVIRONMENTAL RNASES)

Question 13

• 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

Answer 13

NESTED PCR

Question 14

TYPE OF NESTED PCR:
- ONE OF EXTERNAL PRIMERS IS USED ON ‘SECOND ROUND’

Answer 14

SEMI - NESTED PCR

Question 15

TYPE OF NESTED PCR:
- DIFFERENT PAIR OF PRIMERS ARE USED

Answer 15

TRADITIONAL NESTED PCR

Question 16

DISADVANTAGE OF NESTED PCR

Answer 16

• INCREASED OF CONTAMINATION
• LABOR - INTENSIVE
• TIME CONSUMING

Question 17

• 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

Answer 17

REAL - TIME PCR

Question 18

REAL - TIME PCR is plotted in?

Answer 18

SIGMOIDAL CURVE
o X AXIS - PCR CYCLES
o Y AXIS- FLUORESCENCE GENERATED

Question 19

REAL - TIME PCR AMPLIFICATION CONTROL:
- cycles 0 - 15
- start of the run, amount of PCR product is LOW → very little fluorescence

Answer 19

BACKGROUND/BASELINE

Question 20

REAL - TIME PCR AMPLIFICATION CONTROL:
- cycles 15 -25
- amount of PCR product DOUBLES for every PCR cycle

Answer 20

EXPONENTIAL PHASE

Question 21

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

Answer 21

PLATEAU

Question 22

• 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

Answer 22

THRESHOLD CYCLE (CT)

Question 23

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

Answer 23

NONSPECIFIC

Question 24

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

Answer 24

TaqMan PROBE

Question 25

INHIBITS FLUORESCENCE WHEN BOUND TO REPORTER

Answer 25

QUENCHER (3’ END)

Question 26

‘YIELDS FLUORESCENCE’ WHEN QUENCHER IS SEPARATED

Answer 26

REPORTER (5’ END)

Question 27

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

Answer 27

MOLECULAR BEACONS

Question 28

0 FOR FAST CYCLING CONDITIONS
- USEFUL IN FURTHER ANALYSIS LIKE CAPILLARY ELECTROPHORESIS

Answer 28

SCORPION-TYPE PRIMERS

Question 29

• USES 2 SPECIFIC PROBES THAT BIND TO ADJACENT TARGETS

Answer 29

FRET (FLUORESCENT RESONANCE ENERGY TRANSFER)

Question 30

(ACCEPTOR/REPORTER FLUOR)

Answer 30

3’ FLUOROPHORE

Question 31

(DONOR FLUOR)

Answer 31

5’ CATALYST

Question 32

DISADVANTAGES REAL-TIME PCR:

Answer 32

o CANNOT DIFFERENTIATE LIVE OR DEAD CELLS
o PRIMER DIMERS MAY INTERFERE WITH DETECTION (FALSE INCREASED)
o COST OF PRIMERS

Question 33

CONVENTIONAL PCR
DATA ANALYSIS:
QUANTIFICATION::
TURNOVER TIME:

Answer 33

• END POINT
• ELECTROPHORESIS
• LONGER ( PCR + ELECTROPHORESIS)

Question 34

REAL - TIME PCR
DATA ANALYSIS:
QUANTIFICATION::
TURNOVER TIME:

Answer 34

• EXPONENTIAL PHASE OF GROWTH
• DYES
• SHORTER (SIMULTANEOUS MEASUREMENT AND AMPLIFICATION

Question 35

• ENHANCEMENT OF PCR PRODUCTS
• ANNEALING TEMPERATURES HIGHER THAN USUAL AND DECREASED 1C EVERY CYCLE/EVERY OTHER CYCLE UNTIL OPTIMAL ANNEALING TEMPERATURE IS REACHED
• MODIFICATIONS:

Answer 35

TOUCHDOWN PCR

Question 36

LOWERS 5C INSTEAD OF 1C

Answer 36

STEPDOWN PCR

Question 37

ADJUSTMENT OF RAMP SPEED AND COOLING RATE OF THERMAL
CYCLERS

Answer 37

SLOWDOWN PCR

Question 38

APPLICATION OF TOUCHDOWN PCR

Answer 38

• GENE CLONING
• SITE - DIRECTED MUTAGENESIS
• DETECTION OF NON - ABUNDANCE TARGETS

Question 39

• METHOD FOR PREVENTION OF MISPRIMING
• REDUCES PREMATURE DNA POLYMERASE ACTIVITY DURING THE INITIAL STAGES OF THE REACTION SET - UP

Answer 39

HOT START PCR

Question 40

APPLICATIONS OF HOT START PCR

Answer 40

• GENE CLONING
• MUTATIONAL ANALYSIS
• TEMPLATE GENERATION FOR SEQUENCING
• DETECTION OF LOW-COPY NUMBER OF TARGETS