Polymerase Chain Reaction and Gel Electrophoresis:

Theory and Application

Kei-Anne Baritugo
Department of Environmental Engineering
Myongji University
MS/PhD Student
Brown, T. A,. Gene Cloning And DNA Analysis. Oxford: Blackwell Science, 2001. Print.

 Paper Review

o PCR
Theory and concepts
Types & Applications
o Gel Electrophoresis
Theory and concepts
Application

Introduction

Contents:

Polymerase Chain Reaction

Polymerase Chain Reaction

Polymerase Chain Reaction

Type of PCR

Modification

Application

Multiple DNA samples are used

Colony PCR

DNA primers are specific to sequence of gene insert so

only gene of interest is amplified

Screening of microbes for correct DNA insert

Two pairs of primers are used

Nested PCR

1st pair amplifies region flanking gene of interest

2nd pair binds to inner region of 1st amplicon

Very low probability of

nonspecific amplification
Amplification of unknown sequence from
a known template

Inverse PCR

Amplification of DNA with only one

known sequence (involves restriction and ligation)

Used in identifying flanking sequence

of genetic inserts
Identification of sites where retroviruses
integrate into genomic DNA

Multiplex PCR

Use of more than one pair of primers

Long PCR

Use of Pfu polymerase (Pyrococcus furiosus)

Generation of a lot of gene of interests from

one genome sample
DNA sequencing of genomes or long DNA

Polymerase Chain Reaction

Type of PCR
Reverse Transcriptase PCR
rtPCR

Modification
Template is RNA > complementary DNA
transcripts by using reverse transcriptase

Application
Gene Expression Profiling
Identification of sequence of an RNA transcript

Qualitative detection of gene expression

Simultaneous amplification and detection or
quantification of DNA target DNA molecule
Real-time PCR
qPCR

+ nonspecific fluorescent dyes that intercalate w/

any dsDNA

Quantification of DNA

+ sequence specific DNA probes made up of

oligonucleotides labelled w/ fluorescent reporter
permitting detection after hybridization
probe binds to complementary sequence

Asymmetric PCR

2 primers are used 100:1 ratio after 20-25 cycles of

amplification one primer is exhausted so single
stranded DNA is produced in next 5-10 cycles

Diagnostic research for infectious disease, cancer

and genetic mutations
Assessment of water quality
Quantification of gene transcription
Clinical quantification and genotyping of
Hepa B virus
Quantification = degree of infection
copy of viral genome / unit of tissue
synthesis of singlestranded DNA
useful for sequencing or DNA probe
preparations

Gel Electrophoresis

Gel Electrophoresis

Review Article:
Alcaligenes faecalis subsp. phenolicus subsp. nov. a
phenol-degrading, denitrifying bacterium isolated
from a graywater bioprocessor
Marc Rehfuss, James Urban
Systematic and Applied Microbiology
28 (2005) 421429

Methodology:

Isolation of Bacterial Strains

Strain JT
isolated from a Johnson
Space Center graywater
bioprocessor

R2

All turbid mixed samples

were then inoculated onto
R2A (Gibco) plates,
incubated at 30 C

YEP (Yeast Extract Peptone)

Successive transfers to new

plates till colonies are
distinguishable

International Space Station

(ISS) designated ISS ersatz
wastewater

Individual colonies were then

selected for identification and
phenotypic analysis and were
maintained on R2A and TSA
slants

Methodology:

Morphological, Physiological & Biochemical Characterization

Confirmation of biolog reactions were

checked with growth or lack of growth on
M9 agar w/ same C-source as Biolog plates

Methodology:

Extraction of DNA and 16S rDNA analysis

Methodology:

Extraction of DNA and 16S rDNA analysis

Results and Discussion

Conclusion