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Fall 2014, Prof. JB Lee
Polymerase Chain Reaction (PCR) and DNA
Sensors
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
2
DNA (Deoxyribonucleic acid)
A (adenine) T (thymine)
G (guanine) C (cytosine)
…-T-T-C-A-… …-A-A-G-T-…
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
3DNA (Deoxyribonucleic acid)
http://www.dnaftb.org/19/animation.html
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Hydrogen bond
Hydrogen bond forms a double-strand DNA from two single-strand DNAs.
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Complementarity of DNA Molecules
A-T/G-C
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
6PCR (polymerase chain reaction)
Specific genetic sequences (DNA or RNA in (+) or (-) sense) can be replicated and amplified for later sequencing or other analysis.
PCR can make billions of copies of a target sequence of DNA in a few hours
PCR was invented in 1984 as a way to make numerous copies of DNA fragments in the laboratory
Its applications are vast and PCR is now an integral part of molecular biology, gene-based disease detection and forensic science
Amplification of specific gene sequences by PCR
94~95°C
40~65°C
At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA
Fall 2014, Prof. JB Lee
Annealing (primers binding) and extension
Annealing Primers bind to the
complimentary sequence on the target DNA
Extension DNA
polymerase catalyzes the extension of the strand, starting at the primers, attaching the appropriate nucleotide (A-T, C-G)
Fall 2014, Prof. JB Lee
DNA replication
Once the DNA strands are separated, DNA polymerase uses each strand as a template to synthesize new strands of DNA with the precise, complementary order of nucleotides.
DNA polymerase Catalyzes the elongation of DNA by adding nucleoside
triphosphates to the 3’ end of the growing strand DNA polymerase can only add nucleotides to 3’ end of growing
strand The two strands of DNA in a double helix are
antiparallel (i.e. they are oriented in opposite directions with one strand oriented from 5’ to 3’ and the other from 3’ to 5’ The 5′-end ("five prime end") designates the end of the DNA or RNA strand that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus.
https://www.youtube.com/watch?v=wCKF-2nqaOc
Fall 2014, Prof. JB Lee
5’-end and 3’-end
Fall 2014, Prof. JB Lee
DNA ligase
Given the antiparallel nature of DNA and the fact that DNA ploymerase can only add nucleotides to the 3’ end, one strand (referred to as the leading strand) of DNA is synthesized continuously and the other strand (referred to as the lagging strand) is synthesized in fragments (called Okazaki fragments) that are joined together by DNA ligase.
Fall 2014, Prof. JB Lee
DNA replication enzymes
Helicase untwists the two parallel DNA strands
Topoisomerase relieves the stress of this twisting
Single-strand binding protein binds to and stabilizes the unpaired DNA strands
Primase starts an RNA chain and creates a primer (a short stretch RNA with an available 3’ end) that DNA polymerase can add nucleotides to during replication
DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand
Fall 2014, Prof. JB Lee
Taq DNA polymerase
Given that PCR involves high temperatures, it is imperative that a heat-stable DNA polymerase be used in the PCR. Most DNA polymerases would denature (and thus
not function properly) at the high temperatures of PCR.
Taq DNA polymerase is thermally-stable which was first purified from the hot springs bacterium in 1965
Fall 2014, Prof. JB Lee
Taq polymerase
Fall 2014, Prof. JB Lee
PCR thermal cycler
The DNA, DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler
PCR thermal cycler
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
17On-chip PCR
Fall 2014, Prof. JB Lee
PCR primers
Primer is an oligonucleotide sequence – will target a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids For example, there is a ¼ chance (4-1) of finding
an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence, etc.
They are manufactured commercially and can be ordered to match any DNA sequence
Fall 2014, Prof. JB Lee
Primers for genetic disease detection
Primers can be created that will only bind and amplify certain alleles of genes or mutations of genes
PCR is used as a part of the diagnostic test for genetic diseasesHIV (Human immunodeficiency virus) Huntington's disease (abnormal body
movements and reduced mental abilities)Cystic fibrosis (severe breathing difficulties)
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
20Affymetrix Gene Chip
http://www.youtube.com/watch?v=V8uNJCO7Qqo
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
21DNA analysis chip
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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On-Chip Electrophoresis
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Electrokinetic pumping
Electrophoresis Separation of biochemical species based on
electrophoretic mobility (mass-to-charge ratio) under the interaction with an electric field
Electroosmosis Motion of electrolytic solutions near a fixed surface
under the interaction with an electric field
Both mechanisms are important in bio separation technologies such as capillary electrophoresis (CE)
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Electrophoresis
A method of running a mixture of molecules through an agarose gel matrix to separate the components. The mixture is loaded into wells in the agarose plate and a current is passed through the gel. The molecules migrate based on the size of the molecule. The molecules (usually proteins) are then exposed to an imaging molecule (usually ethidium bromide) and are viewed under ultra-violet light.
http://www.youtube.com/watch?v=RNQr7y58QAo
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Elelctroosmosis
Biomedical Applications of Electrical Engineering, Dr. J-B. Lee
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Types of electrophoresis
Southern blot DNA is extracted from cells, and is loaded into wells and run like a gel
electrophoresis, in solutions that are optimized for DNA. The DNA is then transferred out of the gel onto a membrane (nitrocellulose or other), radioactive DNA is then added, and the activity is read or staining is used.
Western blot It is similar to the southern blot except the fact that it is optimized for
protein. The protein is then transferred out of the gel onto a membrane (nitrocellulose or other), antibodies to protein are then added, then another antibody that is conjugated with a radioactive or fluorescent molecule is added and the activity is read with X-ray or radioactivity detector or staining is used.
Northern blot It is similar to the southern blot except the fact that it is optimized for
RNA. The RNA is then transferred out of the gel onto a membrane (nitrocellulose or other), and probed with radioactive RNA or DNA and the activity is read or staining is used.
Sir Edwin Southern