PREFI LAB TOPIC 2 – PCR MODIFICATIONS AND ANALYSIS

MOLECULAR BIOLOGY I PROF EARN IAN CARO, RMT I MARCH 20, 2023

MODIFIED PCR create strand to replace the RNA 

 Adapted for various applications cDNA (a dsDNA) will be amplified
 Types:
o Multiplex PCR
o Sequence specific PCR
o Reverse transcriptase PCR
o Nested PCR
o Real-time PCR
 Other techniques

Multiplex PCR
 Primers can be multiplied (more than a pair).
 Application:
o Multiple amplified targets for human/
pathogen/ forensic:
 Typing
 Identification analyses

TYPES OF REVERSE TRANSCRIPTASE PCR

 Uses Tth polymerase
One-step  RNA sample is directly applied
 Includes initial incubation for RT
(45-50C for 30 minutes)
Two-step  cDNA synthesis  PCR

 Disadvantages: primers may exhibit self-

inhibition (interfere with each other).

Sequence Specific PCR

 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.

 Primers
o Standard Amplification Primers
o Reverse Transcriptase Primers
 Can bind to mRNA’s random sequences
Hexameres/ decameres: 6 or 10
base long primers
Reverse Transcriptase (RT) PCR
 Starting material is RNA
o Reverse transcriptase
 From RNA viruses
 Copies RNA into hybrid RNA-DNA then
uses hairpin formation to DNA stand 

MATEO, JENDY ROMAE EHILLA I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.
PREFI LAB TOPIC 2 – PCR MODIFICATIONS AND ANALYSIS
MOLECULAR BIOLOGY I PROF EARN IAN CARO, RMT I MARCH 20, 2023

 Can bind to mRNA with Poly A tails

18 base long primers

 Applications
o Gold standard for detection of SARS-COV
1/ COVID-19 diagnosis
 Detected 1-3 days before onset
symptoms
 Symptomatic: 1-2 weeks

Real-time PCR
 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.
 Disadvantages
o RNA is vulnerable to degradation
(environmental RNAses)
 Strict specimen handling
 Fixed tissues are difficult to analyze

Nested PCR
 Increased sensitivity and specify of the reaction.
 Uses two pairs of primers for single target.
 Second pair will bind slightly inside the binding
sites of first pair.

 Plotted in sigmoidal curve

o X axis – PCR cycles
o Y axis – fluorescence generated

 Types
o Semi-nested
 One of external primers is used on
second round
o Nested/ traditional
 Different pair of primers are used

MATEO, JENDY ROMAE EHILLA I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.
PREFI LAB TOPIC 2 – PCR MODIFICATIONS AND ANALYSIS
MOLECULAR BIOLOGY I PROF EARN IAN CARO, RMT I MARCH 20, 2023
 Interpretation:
o Fluorescence in earlier cycles  high
amounts of template (target DNA)
o Fluorescence in later cycles  low amount
of template
 Threshold cycle
o CT
o Sample fluorescence crosses the threshold
o Once stablished, starting amount will be
determined
o Applies to qPCR, and RT-qPCR
 Fluorescent and dye-binding regions of DNA are
used:
Nonspecific
 Bind to nonspecific regions of DNA.
 More copies, more fluorescence.
 Requires to prevent mispriming to prevent
fluorescence of those
o EtBr – more toxic
o SYBR Green – reduce toxicity
TaqMan Probe
 Hybridize to sequences between primer and DNA
template
 Exploits the Taq polymerase’s exonuclease
activity (5’-3’)
 Generated by separation of 2 units:
o Quencher – inhibits fluorescence when
bound to reporter
o Reported – yields fluorescence when
quencher is separated
MATEO, JENDY ROMAE EHILLA I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.
 Reporter dyes:
o FAM (6-carboxyfluorescenein)
o TET (6-tetrachlorofluorescenein)
o HEX (6-hexachlorofluorescenein)
o JOE (4’, 5’-dichloro-1’, 7’ -dimethoxy-
fluorrescein)
o Cy3 and Cy5 (indocarbocyanine)
 Quenchers:
o Fluorescent
 DABCYL (4-
dimethylaminophenylazobenzoic
aicd)
 TAMRA ([56]-
carboxytetramethylrhodamine)
o Nonfluorescent
 BH01, BHO2 (black hole quenchers)
 Eclipse
Molecular Beacons
 Measures accumulation of product at annealing
step in the PCR cycle
 25 bases long
 Hairpin/ stem and loop appearance
 Hybridization promotes opening and activation
 Taq polymerase displacement will restore the
hairpin
 Scorpion-type primers
o For fast cycling conditions
o Useful in further analysis like capillary
electrophoresis
FRET (Fluorescent Resonance Energy
Transfer)
 Uses 2 specific probes that bind to adjacent
targets
o 3’ fluorophore (acceptor/ reporter fluor)
o 5’ catalyst (donor fluor)
 Example pairs
o Fluorescein-rhodamine
PREFI LAB TOPIC 2 – PCR MODIFICATIONS AND ANALYSIS
MOLECULAR BIOLOGY I PROF EARN IAN CARO, RMT I MARCH 20, 2023

o Fluorescein-(2 aminopurine)
o Fluorescein-Cy5
 Excitation is transferred from donor to acceptor
and acceptor loses energy in form of heat or
fluorescence

 Disadvantages of Real-time PCR

o Cannot differentiate live or dead cells
o Primer dimers may interfere with detection
(false increased)
o Cost of primers

COMPARISON: CONVENTIONAL VS REAL-TIME PCR

PARAMETER CONVENTIONAL PCR REAL TIME PCT
DATA ANALYSIS Endpoint Exponential phase of
growth
QUANTIFICATION Electrophoresis Dyes
Shorter
TURNOVER TIME Longer (PCR + (simultaneous
Electrophoresis measurement and
amplification)

Other techniques

Touchdown PCR
 Enhancement of PCR products
 Annealing temperatures higher than usual and
decreased 1C every cycle/ every other cycle until
optimal annealing temperature is reached
 Modifications:
o Stepdown PCR – lowers 5C instead of 1C
o Slowdown PCR – adjustment of ramp
speed and cooling rate of thermal cyclers

Hot start PCR

 Method for prevention of mispriming.

MATEO, JENDY ROMAE EHILLA I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.