MOLECULAR BIOLOGY
Transcribed by: Rainiel Crista N. Sorra | MIDTERMS
CHAPTER V: AMPLIFICATION • Allows the production of millions of copies of DNA
in 1-2 hours
TECHNIQUES - PCR Using the following components:
o Template
Topic Outline:
• Amplification Techniques o DNA polymerase
o 3 general types o dNTPs (bases)
• PCR-Target Amplification o primers
o Steps in PCR
o Components of PCR Amplicons – the copies produced in PCR
• Detection of Amplicons
STEPS IN PCR
AMPLIFICATION TECHNIQUES 1. DENATURATION – temperature 94-96 degrees
• Molecular diagnostics made diagnosis of Celsius for 3-5 minutes
diseases using nucleic acid samples - Separation of double stranded DNA
• Limited concentration of nucleic acid >> false 2. ANNEALING – temperature 50-70 degrees
negative detection Celsius for 10-30 seconds
- The primers hybridize or attaches to a
3 GENERAL TYPES OF AMPLIFICATION specific part of the template
3. EXTENSION – temperature 68-72 degrees
TECHNIQUES
Celsius for 5-10 minutes
1. Target Amplification – target sequence is well - DNA polymerase synthesizes a copy of DNA
defined and is copied many times in vitro template by adding nucleotide bases to the
Examples: hybridized primers
o PCR
o TMA
o LAMP
o WGA
2. Signal Amplification – multiplying & amplifying
the signal produced while maintaining the amount
of the target
Examples:
o Branched DNA (bDNA)
o Serial invasive signal amplification
3. Probe Amplification – the probe or product of a
probe that is attached to the target is amplified
o Rolling Circle Amplification (RCA)
o Ligase Displacement Amplification
PCR- TARGET AMPLIFICATION
PCR
• Originally called polymerase-catalyzed chain
reaction, was 1st discovered in 1983 by Kary
Mullis
• The 1st successful amplification was made in a
short fragment of E. coli plasmid, pBR322
• The 1st clinical application was the amplification
of beta globin for diagnosis of sickle cell anemia
• Described as in vitro replication of DNA
RCNS | 1
COMPONENTS OF PCR
• DNA Template – obtained from
genomic/mitochondrial DNA of human or
microorganisms
• Primers – are oligonucleotides; short, single-
stranded DNA sequence; 18-25 base pairs long
each
• Polymerase Enzyme
o Tag DNA polymerase – most common
replication enzyme used for PCR
o Isolated from extremophile Thermus
aquaticus and a heat-resistant enzyme
with a half-life of 40 minutes at 95
degrees Celsius (optimal temp is 72
degrees Celsius)
Other PCR enzymes include:
• Pwo DNA polymerase – cloned from
hyperthermophilic bacterium Pyrococcus woesei
• Tth DNA polymerase – isolated from Thermus
thermophilus spp.
*Recommended polymerase concentration is usually 1-2
5 units per 100 uL reaction volume
• Nucleotide Bases
BUFFERS
• pH 8.0-9.5
Contains: potassium ions or ammonium sulfate
DETECTION OF AMPLICONS
• Agarose Gel Electrophoresis
o Agarose gel is easy and cost effective
medium
• Polyacrylamide Gel-electrophoresis (PAGE)
o Visualization of PCR product
o Used for separation of DNA molecules
o Much more difficult and time consuming
that agarose gel
VISUALIZATION
Ethidium Bromide Staining – staining with ethidium
bromide + visualizing the gel on UV trans-illuminator is the
most common and least expensive method
