Polymerase Chain Reaction (PCR) and DNA Sensors

1. Polymerase Chain Reaction (PCR) and DNA Sensors

2. DNA (Deoxyribonucleic acid) A (adenine) T (thymine) G (guanine) C (cytosine) …-T-T-C-A-… …-A-A-G-T-…

3. DNA (Deoxyribonucleic acid) http://www.dnaftb.org/19/animation.html

4. Hydrogen bond Hydrogen bond forms a double-strand DNA from two single-strand DNAs.

5. Complementarity of DNA Molecules A-T/G-C

6. PCR (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

7. Amplification of specific gene sequences by PCR

8. 94~95°C At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA 40~65°C

9. 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)

10. DNA replication https://www.youtube.com/watch?v=wCKF-2nqaOc • 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.

11. 5’-end and 3’-end

12. DNA ligase • Given the antiparallel nature of DNA and the fact that DNA ploymerasecan 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.

13. DNA replication enzymes • Helicase untwists the two parallel DNA strands • Topoisomeraserelieves 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

14. 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. • TaqDNA polymerase is thermally-stable which was first purified from the hot springs bacterium in 1965

15. Taq polymerase

16. 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

17. On-chip PCR

18. 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

19. 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 diseases • HIV (Human immunodeficiency virus) • Huntington's disease (abnormal body movements and reduced mental abilities) • Cystic fibrosis (severe breathing difficulties)

20. Affymetrix Gene Chip http://www.youtube.com/watch?v=V8uNJCO7Qqo

21. DNA analysis chip

22. On-Chip Electrophoresis

23. 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)

24. 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

25. Elelctroosmosis

26. 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