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Chapter 5 Molbio

The document discusses molecular biology techniques, specifically focusing on DNA amplification methods such as PCR, which allows for the rapid production of millions of DNA copies. It outlines the steps involved in PCR, including denaturation, annealing, and extension, as well as the necessary components like DNA template, primers, and polymerase enzymes. Additionally, it covers the detection of amplicons through methods like agarose gel electrophoresis and the visualization of results.

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Rainiel Crista Sorra
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9 views

Chapter 5 Molbio

The document discusses molecular biology techniques, specifically focusing on DNA amplification methods such as PCR, which allows for the rapid production of millions of DNA copies. It outlines the steps involved in PCR, including denaturation, annealing, and extension, as well as the necessary components like DNA template, primers, and polymerase enzymes. Additionally, it covers the detection of amplicons through methods like agarose gel electrophoresis and the visualization of results.

Uploaded by

Rainiel Crista Sorra
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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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

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