Common Molecular Biology Techniques and Their Uses in Chemistry Becky Farmer Long Literature Seminar August 25, 2008 What is Molecular Biology? •Molecular biology is commonly defined as the branch of biology that examines biological phenomena at the molecular level •Mainly concerned with the study of biological macromolecules, such as DNA, RNA and proteins and their various interactions •Term “molecular biology” was coined in 1938 by Warren Weaver, the former director of the Division of Natural Sciences at the Rockefeller Foundation •Characterized by several common techniques: •Polymerase chain reaction (PCR) •Polyacrylamide gel electophoresis and blotting •Fluorescence activated cell sorting (FACS) •Knockout mice •RNA interference (RNAi) •Microarrays •Confocal microscopy •Fluorescent in situ hybridization (FISH) •Expression cloning/cell transfection •And many others….. The Basics: DNA, RNA and Everything In Between •The process by which genetic information is shared and expressed in the cell is known as the “central dogma of molecular biology” •DNA RNA protein •DNA is duplicated through the process of replication •mRNA is made through transcription •The genetic code is read from the mRNA by ribosomes, which synthesize proteins through the process of translation The Basics: DNA Replication •Genetic information is passed on to new cells via the process of semi-conservative replication •Each DNA strand containing the genetic code is copied by DNA polymerase III in a 5’3’ fashion .The leading strand is copied continuously, while the lagging strand is copied in short pieces (Okazaki fragments) and then ligated together •DNA helicase unwinds the leading edge of the replication fork, while topoisomerase prevents tangling of the chromosomal material Polymerase Chain Reaction (PCR) •Developed in 1983 by Kary Mullis, who received a Ph.D. in biochemistry from the University of California at Berkeley •Mullis conceived of the idea for PCR while working for Cetus Corporation, a biotechnology company located in Emeryville, CA •Mullis received the Nobel Prize in Chemistry for his work in 1993, the first prize ever awarded for research performed at a biotechnology company •In the press release for the Nobel Prize, the Royal Swedish Academy of Sciences described the impact of PCR as follows: Kary Mullis The biomedical applications of the PCR method are already legion. Now that it is possible to discover very small amounts of foreign DNA in an organism, viral and bacterial infections can be diagnosed without the time-consuming culture of microorganisms from patient samples. PCR is now being used, for example, to discover HIV infections. The method can also be exploited to localize the genetic alterations underlying hereditary diseases. Thus PCR, like site-directed mutagenesis, has a great potential within gene therapy. […] In police investigations PCR can give decisive information since it is now possible to analyze the DNA in a single drop of blood or in a hair found at the scene of a crime. Mechanism of PCR •PCR is used to exponentially amplify a sequence of DNA, known as the target sequence •Isolated DNA is added to reaction along with primers (complementary to flanking regions of target sequence), dNTPs, and heat-stable DNA Taq polymerase •First step involves heating the reaction to ~95 °C for 20-30 seconds to denature the double helix •Rapid cooling to 50-65 °C allows for annealing of primers •Reaction is then heated to 72 °C, the optimal temperate for the Taq polymerase, which then synthesizes new DNA strands from primers in 5’3’ direction Mechanism of PCR Continued •Another cycle then proceeds with the same temperature changes, or “steps”, with the newly synthesized DNA strands acting as templates •After the second cycle, short strands containing only the DNA sequence of interest are formed •These short strands can act as templates for further cycles, thereby leading to exponential amplification of the sequence of interest •Consists of generally 20-40 cycles, with each cycle consisting of discrete temperature steps •Ideal situation is amplification of 2n with n equal to the number of cycles .Amplification is a millionfold after 20 cycles and a billionfold after 30 cycles Quantitative Real-Time PCR (qRT-PCR) •Quantitation of gene expression levels is important for understanding of cell biology •Use of fluorescence emission generated during each cycle allows accurate quantitation of gene of interest •Most commonly used method involves fluorescent probe that hybridizes to sequence of interest •When Taq polymerase reaches probe, 5’ 3’ exonuclease activity destroys the probe and releases fluorescent dye Mocellin, S.; Rossi, C. R.; Pilati, P.; Nitti, D.; Marincola, F. M. Trends Mol. Med. 2003, 9, 189-195. The Basics: Transcription •Transcription is the process by which the genetic code in DNA is passed on to single- stranded mRNA .Particularly important step in gene expression since this is the level at which the cell regulates which proteins are made •RNA polymerase (I, II or III) binds to a promoter in the DNA sequence with the assistance of multiple transcription factors and synthesizes the gene .Promoter usually 25 nucleotides upstream of the gene of interest •DNA helix reassembles after transcription, while mRNA is extruded from the nucleus, post-transcriptionally modified and translated into protein Reverse Transcription PCR (RT-PCR) •Reverse transcription is the process used by viruses to synthesize DNA from RNA genome •Similar principle is used for reverse transcription PCR to convert RNA to cDNA •Technique used for measurement of gene expression in the cell •Often combined with quantitative real-time PCR for accurate picture of actions occurring inside cell Bustin, S. A.; Mueller, R. Clin. Sci. 2005, 109, 365-379. Electrochemical Real-Time PCR •Current real-time PCR protocols rely on bulky optical systems for detection of amplification •For point-of-care applications, need an inexpensive portable device •In the first step, PCR is carried out in solution phase to give dsDNA containing modified Fc markers •The DNA is then annealed to an electrode containing complementary probes •Extension occurs, increasing number of Fc markers on surface electrical signal Yeung, S. S. W.; Lee, T. M. H.; Hsing, I. J. Am. Chem. Soc. 2006, 128, 13374-13375. Electrochemical Real-Time PCR Continued •Voltammetric scans taken in presence (sample) and absence (negative control) of target sequence demonstrate the buildup of Fc markers on glass electrode •Current measured as a function of cycle number shows that signal is detected on a real-time basis •Signal is detected more quickly than in traditional optical systems could be used for detection of small amounts of DNA Yeung, S. S. W.; Lee, T. M. H.; Hsing, I. J. Am. Chem. Soc. 2006, 128, 13374-13375. Polyacrylamide Gel Electrophoresis (PAGE) •Gel electrophoresis is used to separate DNA, RNA or proteins by size and charge •The gel is made of a crosslinked polyacrylamide, although it can also be made of agarose •Current is applied to the gel, which causes the proteins/nucleic acids of interest to migrate toward anode/cathode causes discrete bands which can be identified by size •SDS-PAGE is used to analyze proteins, since sodium dodecyl sulfate denatures proteins Blotting •Gel electrophoresis is the first step in the biological procedure known as blotting •Once macromolecules of interest have been separated using gel electrophoresis, they are transferred to a separate surface where they are further analyzed Edwin Southern •Types of blots: .Southern blot – used to probe for a specific sequence of DNA, named after Edwin Southern, a British biologist who developed the technique in 1975 .Western blot – analysis of proteins via antibody detection .Northern blot – detection of specific sequences of RNA The Basics: Translation •Translation is the process by which the genetic code found in mRNA is used to synthesize proteins •Codons (sequence of three nucleotides) are matched to their corresponding tRNA carrying the appropriate amino acid •The ribosome catalyzes the elongation of the growing peptide chain and then transports the nascent protein into the endoplasmic reticulum, where it is folded into its 3D structure and post-translationally modified Western Blots •Western blots are the most commonly used blotting technique in molecular biology •Used to gauge the level of protein expression in the cell since protein levels are a result of gene expression, Western blot gives accurate depiction of gene activity in the cell •First developed by George Stark at Stanford University, then later perfected and termed “western blot” by W. Neal Burnette in 1981 •Western blotting is used to diagnose several diseases, including HIV and bovine spongiform encephalopathy Western Blots in Hsp90 Inhibitor Evaluation •Hsp90 is one of the most abundant proteins in humans accounts for 1-2% of all protein •Assists in protein folding as a chaperone and is also important in tumor progression .Stabilizes mutant oncogenic proteins such as p53 and bcr/abl .Maintains PI3K and AKT, both of which inhibit apoptosis .Promotes metastasis (MMP-2) and angiogenesis (NOS and VEGF) Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-12779. Hsp90 Inhibitor Development Continued •Each compound was tested for its ability to cause degradation of Hsp90 client protein phospho-AKT in SKBR3 breast cancer cells •Geldanamycin was used as positive control •SAR shows several patterns: .7-position of coumarin ring is important for activity (B vs. D/E) .Compounds with amide linker show more potent action (A) .Diol may mimic ATP in binding site (4) •Compound A4 was identified as the most potent inhibitor Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-12779. Hsp90 Inhibitor Development Continued •Novobiocin analog A4 also tested to determine their action against hormone receptors •Two cell lines were used: LNCap (mutated androgen receptor PCa) and LAPC-4 (wild-type androgen receptor PCa) •A4 causes degradation of androgen receptor (AR), AKT and HIF-1α at low concentrations (~1µM) Yu, X.