PCR, real time PCR, DNA sequencing, Protein Electrophoresis, DNA Microarray.
Polymerase Chain Reaction
Enables researchers to quickly make a specific DNA without building and screening a library.
Has three major steps (called cycles), repeated about twenty-five to forty times:
1. Denaturing of DNA by high temperatures.
2. Attachment of single-stranded DNA primers that flank the desired DNA at a
lower temperature.
3. DNA is synthesized by a special heat-stable DNA polymerase called “Taq DNA
polymerase” and isolated from the thermophilic bacterium Thermus
aquaticus.
Polymerase chain reaction is a powerful method for multiplying specific sequences of DNA.
Short fragments of DNA called oligonucleotides act as primers to allow a specific DNA to be
replicated by DNA polymerase.
Video: 19-polymerase-chain-reaction
http://www.dnalc.org/files/videos/dnai/mp4/19-polymerase-chain-reaction.mp4
Animation: pcr
http://www.dnalc.org/resources/animations/pcr.html
Video: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html#
PCR is used to:
1.
Isolate specific sequences for further study.
2.
Identify specific genetic loci for diagnostic or medical purposes.
3.
Generate DNA fingerprints to determine genetic relationships or in forensics.
4.
Provide specific DNA for rapid sequencing.
89
Real-Time qRT-PCR
(Real-Time Quantitative Reverse Transcription PCR) is a major development of PCR
technology that enables reliable detection and measurement of products generated during
each cycle of PCR process. This technique became possible after introduction of an
oligonucleotide probe which was designed to hybridize within the target sequence.
Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can
be used to detect amplification of the target-specific product.
90
TaqMan assay (named after
Taq DNA polymerase) was
one of the earliest methods
introduced for real time PCR
reaction monitoring and has
been widely adopted for
both the quantification of
mRNAs and for detecting
variation. The method
exploits the 5' endonuclease
activity of Taq DNA
polymerase to cleave an
oligonucleotide probe
during PCR, thereby
generating a detectable
signal. The probes are
fluorescently labeled at their
5' end and are nonextendable at their 3' end by
chemical modification.
Specificity is conferred at
three levels: via two PCR
primers and the probe.
Applications of Real Time Quantitative RT-PCR




Relative and absolute quantification of gene expression.
Validation of DNA microarray results.
Variation analysis including SNP discovery and validation.
Counting bacterial, viral, or fungal loads, etc.
91
Sanger Sequencing of DNA
A DNA primer is annealed to the desired DNA.
DNA polymerase extends to primer, and labeled nucleotides incorporate in the newly
made DNA.
2’, 3’-dideoxynucleotides are incorporated and stop DNA synthesis in four test tubes, with
each tube containing one dideoxynucleotide such as ddATP, ddGTP, ddCTP, and ddTTP.
Each resulting strand is a different length, and is separated by electrophoresis.
Video: 29-sanger-sequencing
http://www.dnalc.org/resources/3d/29-sanger-sequencing.html
Video: sangerseq
http://www.dnalc.org/resources/animations/sangerseq.html
Video: cycseq
http://www.dnalc.org/resources/animations/cycseq.html
The structure of a dideoxynucleotide shows the substitution of an H instead of an OH on
the 3’ carbon of the deoxyribose.
92
Automating Sanger Sequencing
Video: 29-sanger-sequencing
http://www.dnalc.org/resources/3d/29-sanger-sequencing.html
Video: sangerseq
http://www.dnalc.org/resources/animations/sangerseq.html
Video: cycseq
http://www.dnalc.org/resources/animations/cycseq.html
DNA sequencing lets us:
1.
Confirm the identity of genes isolated from DNA libraries.
2.
Find the DNA sequence of regulatory DNA elements such as promoters.
3.
Reveal the fine structure of genes and other DNA.
4.
Confirm the DNA sequence of cDNA and other DNA made in the laboratory.
5.
Determine the amino acid sequence of a gene or cDNA from the DNA sequence.
93
Protein Methods
Protein Gel Electrophoresis can be used to separate individual proteins by size or charge.
One-dimensional electrophoresis uses polyacrylamide gel (PAGE).
It gives bands of protein.
Two-dimensional electrophoresis has two steps.
a)
Isoelectric focusing separates proteins in a pH gradient. The proteins move until they
have no charge. This is called the “isoelectric point.”
b)
This gel is then put on another gel which has sodium dodecyl sulfate (SDS-PAGE). SDS
gives proteins a negative charge. The electrophoresis separates the protein by size.
Video Protein Electrophoresis:
http://highered.mheducation.com/sites/9834092339/student_view0/chapter20/electroph
oresis.html
Protein engineering changes the protein’s gene sequence to change the amino acid
sequence, and so change the protein’s properties. It may make the protein function better
or worse.
DNA Microarray Technology
Uses hybridization.
It can be used to:
1.
Analyze gene activity, ie gene expression (transcription).
2.
Follow changes in genomic DNA.
Each spot on the slide or chip represents the location of many copies of a signal DNA
sequence.
94
The steps in preparing a microarray, preparing and hybridizing labeled target cDNA, and
analyzing the data.
Video 26-microarray:
http://www.dnalc.org/resources/3d/26-microarray.html
Microarrays:
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/mic
ro50.swf::Microarray
Dnaarray:
http://www.dnalc.org/resources/animations/dnaarray.html
Dnachip:
http://www.dnalc.org/resources/animations/dnachip.html
95