* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Recombinant DNA Technology
Whole genome sequencing wikipedia , lookup
History of RNA biology wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Genetic engineering wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Nutriepigenomics wikipedia , lookup
DNA profiling wikipedia , lookup
Human genome wikipedia , lookup
Designer baby wikipedia , lookup
Cancer epigenetics wikipedia , lookup
DNA sequencing wikipedia , lookup
DNA polymerase wikipedia , lookup
Point mutation wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Microevolution wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Metagenomics wikipedia , lookup
DNA vaccination wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genome editing wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Microsatellite wikipedia , lookup
DNA supercoil wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Epigenomics wikipedia , lookup
Molecular cloning wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Genomic library wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Primary transcript wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
History of genetic engineering wikipedia , lookup
Helitron (biology) wikipedia , lookup
Recombinant DNA Technology Isolating DNA • Chemically, DNA is a very simple compound, with little variation • between species. The basic steps: 1. 2. 3. 4. • • Methods for breaking the cells open vary between species, but usually involve mechanical disruption in a buffer that inhibits DNAases. Separating DNA from other cell components relies on the fact that DNA is chemically different from proteins and other macromolecules. – – • Break the cells open Disrupt cell membranes with a detergent Remove proteins and other macromolecules Concentrate the DNA by precipitating it and re-suspending it in fresh buffer. If the cell extract is mixed with a phenol/chloroform solution, most of the proteins and other cellular junk goes into the phenol/chloroform layer while the DNA and RNA stay in the aqueous phase. Phenol is very nasty, and many methods have been invented to get around this step. DNA can be precipitated using ethanol, and then resuspended in a buffer containing EDTA, which chelates (removes from solution) Mg2+ ions. This is useful because all DNA-degrading enzymes use Mg2+ ions as a co-factor. Transformation • How to get DNA back into the cell after manipulation in vitro. – – – • • • • Transformation means the cell takes up naked DNA from the environment and incorporates it into its genome. In animal cells, this process is often called transfection, because transformation also means going from normal to cancerous. A cell that is in a state that allows transformation is called competent. Need to get the DNA through the cell membrane, plus possibly the cell wall (bacteria and plants) and get it into the nucleus (eukaryotes). Lots of methods, many are specific to a particular group of organisms. Natural competence: some bacteria take DNA up without any special treatment. Good example: Streptococcus pneumoniae, used by Griffith, and Avery, MacLeod and McCarty in early experiments showing that DNA with the hereditary material. Chemically induced competence: E. coli is made competent by treating the cells with Ca2+ ions, then giving them a heat shock (2 minutes at 42oC). More Transformation • Electroporation: if you subject a cell to a high voltage electrical field (say 20,000 volts per centimeter), holes appear in the membrane that DNA can go through. The holes disappear quickly once the voltage disappears. • Gene gun: Tiny gold particles are coated with DNA, then blasted through the cell wall using high pressure gas. One method uses blank .22 rifle cartridges to generate the pressure. • Lipofection: coating the DNA in lipid vesicles that fuse with the cell membrane. This is very efficient and common with mammalian cells. Electrophoresis • • • • • • Electrophoresis is the separation of charged molecules in an electric field. Nucleic acids have 1 charged phosphate (- charge) per nucleotide. This implies a constant charge to mass ratio. Thus, separation is based almost entirely on length: longer molecules move slower. Done in a gel matrix to stabilize: agarose or acrylamide. average run: 100 Volts across a 10 cm gel, run for 2 hours. Stain with ethidium bromide: intercalates between DNA bases and fluoresces orange with UV light. Run alongside standards of known sizes to get lengths Restriction Enzymes • Restriction enzymes (or more accurately, restriction endonucleases) are used by bacteria to destroy invading DNA. • • • • The enzymes recognize particular sequences in DNA and then cut the DNA. Their own DNA has been modified (methylated) at the corresponding sequences by a restriction methylase (modification enzyme), so only foreign DNA is cut. There are several different types of restriction enzyme: type I, type II, etc.. They differ on where the cut the DNA relative to the recognition site: some cut randomly up to 1000 bp from the recognition site. In molecular biology we use type II restriction enzymes, which cut the DNA at the recognition site Restriction Sites • Most restriction sites are palindromes: they read the same forwards and backwards (remembering that “reading backwards” means reading on the other strand). – For example, Eco R1 cuts at GAATTC. TTC is what is on the opposite strand of GAA, read 5’ to 3’. • Many restriction enzymes give staggered cuts: which leave a few bases at the ends single stranded. – This is very useful for cloning: staggered cut sites are sticky: the unpaired bases pair with unpaired bases on another DNA molecule, holding the two molecules together long enough for DNA ligase to attach them covalently. – An enzyme that cuts both strands in the same place (e.g. Alu1) produces blunt ends. Restriction Enzymes for Mapping • Most restriction sites are 4 bp or 6 bp long. – • The locations of the sites are fixed points that are easy to detect, so they are good for mapping. – • In random DNA, a 4 bp site occurs about every 44 = 256 bp, and a 6 bp site appears about every 46 = 4096 bp. Restriction sites near genes can be used for cutting out a gene or splicing something in. Basic technique: – – – Digest DNA with each restriction enzyme individually and in pairs Measure band sizes using electrophoresis Assemble a map from the band sizes. Single digests tell you how many sites each enzyme has and how far apart they are. Double digests allow you to put the sites in the proper order. RNA • Gene expression is studied using RNA. However, RNA has two annoying properties: – it is very easily degraded. A desirable property in the cell: allows rapid response to environmental changes – It usually has a lot of secondary structure. This means that migration speed in electrophoresis is not proportional to length. The same problem occurs with proteins. • For direct analysis of RNA, denaturing agents such as formaldehyde or methyl mercury hydroxide (very toxic) are used. • For sequence analysis, RNA is first converted to DNA (called cDNA). cDNA Synthesis •use oligo-dT primer, which binds to poly-A tail. •make the first DNA strand from the RNA using reverse transcriptase, which makes a DNA copy of an RNA molecule. More cDNA Synthesis •Remove the RNA with heat or alkali. •The 3’ end spontaneously forms a small hairpin. •Extend the hairpin with DNA polymerase •Cut the loop with S1 nuclease (which cuts at unpaired bases) •Attach synthetic linkers with DNA ligase and clone into a vector. Expressed Sequence Tags • ESTs are cDNA clones that have has a single round of sequencing done from one end. • First extract mRNA from a given tissue and/or environmental condition. Then convert it to cDNA and clone. • Sequence thousands of EST clones and save the results in a database. • A search can then show whether your sequence was expressed in that tissue. – quantitation issues: some mRNAs are present in much higher concentration than others. Many EST libraries are “normalized” by removing duplicate sequences. • Also can get data on transcription start sites and exon/intron boundaries by comparing to genomic DNA – but sometimes need to obtain the clone and sequence the rest of it yourself. Finding Genes • How to find one gene in large genome? A gene might be 1/1,000,000 of the genome. Three basic approaches: • 1. Polymerase chain reaction (PCR). Make many copies of a specific region of the DNA. • 2. cell-based molecular cloning: create and isolate a bacterial strain that replicates a copy of your gene. • 3. hybridization: make DNA single stranded, allow double strands to re-form using a labeled (e.g. radioactive) version of your gene to make it easy to detect. Polymerase Chain Reaction • Based on DNA polymerase creating a second strand of DNA. – Needs template DNA and two primers that flank the region to be amplified. Primers are short (generally 18-30 bases) DNA oligonucleotides complementary to the ends of the region being amplified. – DNA polymerase adds new bases to the 3' ends of the primers to create the new second strand. – go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA from this region – A key element in PCR is a special form of DNA polymerase from Thermus aquaticus, a bacterium that lives in nearly boiling water in the Yellowstone National Park hot springs. This enzyme, Taq polymerase, can withstand the temperature cycle of PCR, which would kill DNA polymerase from E. coli. • advantages: – rapid, sensitive, lots of useful variations, robust (works even with partly degraded DNA) • disadvantages: – Only short regions (up to 2 kbp) can be amplified. – limited amount of product made PCR Cycle • • • • PCR is based on a cycle of 3 steps that occur at different temperatures. Each cycle doubles the number of DNA molecules: 25-35 cycles produces enough DNA to see on an electrophoresis gel. Each step takes about 1 minute to complete. 1. Denaturation: make the DNA single stranded by heating to 94oC 2. Annealing: hybridize the primers to the single strands. Temperature varies with primer, around 50oC 3. Extension: build the second strands with DNA polymerase and dNTPs: 72oC. Other PCR Images SSR Genetic Markers • . Microsatellites (also called Simple Sequence Repeats (SSRs) or Short Tandem Repeats (STRs). – – • SSRs are short (2-5 bases) sequences that are repeated several times in tandem: TGTGTGTGTGTG is 6 tandem repeats of TG. – – – • • Used for genome mapping in humans, plants, and other organisms. Also used for forensic DNA analysis (criminal identification) SSRs are found in and near many genes throughout the genome--they are quite common and easy to find. During normal replication of the DNA in the nucleus, DNA polymerase sometimes slips and creates extra copies or deletes a few copies of the repeat. This happens rarely enough that most people inherit the same number of repeats that their parents had (i.e. SSRs are stable genetic markers), but often enough that numerous variant alleles exist in the population. SSRs are detected using PCR. A pair of primers binds to sites flanking the SSR locus. This region is amplified by PCR, and the products are examined on an electrophoresis gel. The FBI uses the Combined DNA Index System (CODIS) in crime investigations. CODIS is a set of 13 microsatellite loci scattered throughout the genome, with many alleles (repeat numbers) for each locus. SSR Example Real Time PCR • Used to quantitate gene expression: • First, convert all mRNA in a sample to single stranded cDNA using reverse transcriptase, Then, amplify the region of interest using specific primers. Measure the amount of DNA made by PCR using a dye that only binds to double stranded DNA. SYBR Green is a commonly used dye. The more mRNA/cDNA you started with, the faster the fluorescence builds up • • • In this RT-PCR experiment, 30, 300, or 3000 copies of the cDNA were subjected to PCR. The more copies of the cDNA, the sooner the fluorescence rises and saturates the detector. Cell-Based Molecular Cloning • The original recombinant DNA technique: 1974 by Cohen and Boyer. • Several key players: • 1. restriction enzymes. Cut DNA at specific sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC. • 2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated. – Plasmids for cloning carry drug resistance genes that are used for selection. – Spread antibiotic resistance genes between bacterial species • • 3. DNA ligase. Attaches 2 pieces of DNA together. 4. transformation: DNA manipulated in vitro can be put back into the living cells by a simple process . – The transformed DNA replicates and expresses its genes. Plasmid Vectors • To replicate, a plasmid must be circular, and it must contain a replicon, a DNA sequence that DNA polymerase will bind to and initiate replication. Also called “ori” (origin of replication). – – • • • Replicons are usually species-specific. Some replicons allow many copies of the plasmid in a cell, while others limit the copy number or one or two. Plasmid cloning vectors must also carry a selectable marker: drug resistance. Transformation is inefficient, so bacteria that aren’t transformed must be killed. Most cloning vectors have a multiple cloning site, a short region of DNA containing many restriction sites close together (also called a polylinker). This allows many different restriction enzymes to be used. Most cloning vectors use a system for detecting the presence of a recombinant insert, usually the blue/white betagalactosidase system. Basic Cloning Process • Plasmid is cut open with a restriction enzyme that leaves an overhang: a sticky end • Foreign DNA is cut with the same enzyme. • The two DNAs are mixed. The sticky ends anneal together, and DNA ligase joins them into one recombinant molecule. • The recombinant plasmids are transformed into E. coli by treating the cells with Ca2+ ions and a heat shock. • Cells carrying the plasmid are selected by adding an antibiotic: the plasmid carries a gene for antibiotic resistance. DNA Ligase in Action! I hope Cloning Vector Types • For different sizes of DNA: – plasmids: up to 5 kb – phage lambda (λ) vectors (also cosmids): up to 50 kb – BAC (bacterial artificial chromosome): 300 kb – YAC (yeast artificial chromosome): 2000 kb • Expression vectors: make RNA and protein from the inserted DNA – shuttle vectors: can grow in two different species, usually E. coli and something else Bacterial Artificial Chromosomes • • • • • Based on the F plasmid that confers the ability to conjugate. Low copy number plasmids (usually 1 per cell), which prevents crossing over between repeated sequences in the insert DNA But, low copy number also means low DNA yield. Transformed into E. coli using electroporation, subjecting the bacteria to a high voltage electrical field. BACs are currently the most common vector for large inserts such as eukaryotic genome projects. Expression Vectors • Various types: – RNA only: use a vector that has a phage T7 promoter in front of the cloning site, and an inducible T7 polymerase gene. Induction by the lac operon repressor gene and the synthetic inducer IPTG (isopropyl thiogalactoside). – polypeptide or fragments of polypeptides: can be produced in E. coli using a ribosome binding site in addition to the promoter. Need to use the correct reading frame. • can also be done as a fusion protein (your protein fused to a marker protein) for easy detection or purification – post-translationally modified or intron-spliced protein: needs to be expressed in eukaryotic cells. Needs eukaryotic promoter and polyadenylation (poly-A addition) signals, plus a selectable marker that works in eukaryotes (since most antibiotics are specific for prokaryotes). Example Expression Vector • • • • • For eukaryotic expression, this vector (from Invitrogen) has a cauliflower mosaic virus promoter (PCMV), a bovine growth hormone polyadenlyation site (BGHpA). The DNA inserted at “hORF” gets fused to a short peptide called an epitope, for which very specific anitbodies exist. It also gets fused to 6 histidines, which allow easy purification on a column that has nickel ions bound to it (an affinity tag). For growth in mammalian cells, it has an SV40 viral origin of replication (SV40ori), and a zeocin resistance gene (Zeocin, with SV40 promoter/enhancer and SV40 poly A site). For growth in E. coli it has the ColE1 replicon. Zeocin works as a selectable marker in bacteria as well as in eukaryotic cells. There is also a T7 promoter for making RNA from the inserted gene, and an f1 origin of replication for making single stranded DNA (useful for sequencing). Hybridization • The idea is that if DNA is made single stranded (melted), it will pair up with another DNA (or RNA) with the complementary sequence. If one of the DNA molecules is labeled, you can detect the hybridization. • Basic applications: – Southern blot: DNA digested by a restriction enzyme then separated on an electrophoresis gel – Northern blot: uses RNA on the gel instead of DNA – in situ hybridization: probing a tissue – colony hybridization: detection of the clone you want from a library – Microarrays: quantitation of all mRNAs in a cell Labeling • Several methods. One is random primers labeling: – use 32P-labeled dNTPs – short random oligonucleotides as primers (made synthetically) – single stranded DNA template (made by melting double stranded DNA by boiling it) – DNA polymerase copies the DNA template, making a new strand that incorporates the label. • Can also label RNA (sometimes called riboprobes), use non-radioactive labels (often a small molecule that labeled antibodies bind to, or a fluorescent tag), use other labeling methods. Hybridization Process • • • • All the DNA must be single stranded (melt at high temp or with NaOH). Occurs in a high salt solution at about 60oC. Complementary DNAs find each other and stick. Need to wash off nonspecific binding. Stringency: how perfectly do the DNA strands have to match in order to stick together? Less than perfect matches will occur at lower stringency (e.g. between species). Increase stringency by increasing temp and decreasing salt concentration. Rate of hybridization depends on DNA concentration and time (Cot), as well as GC content and DNA strand length. Autoradiography. Put the labeled DNA next to X-ray film; the radiation fogs the film. Southern Blot • • • • • • Used to detect a specific DNA sequence in a complex mixture, such as genomic DNA Cut DNA with restriction enzyme, then run on an electrophoresis gel. Suck buffer through the gel into a nitrocellulose membrane. The buffer goes through but the DNA sticks to the membrane. Fix the DNA to the membrane permanently with UV or heat Hybridize membrane to a radioactive probe, then detect specific bands with autoradiography. Northern blot uses RNA instead. RNA must be denatured so the distance it migrates on the gel is proportional to its length: put formaldehyde in the gel. In Situ Hybridization • Using tissues or tissue sections. • Often done with nonradioactive probes because the high energy of 32P emission gives an imprecise view of where the hybridization is. • Counterstain the tissue so non-hybridizing parts are visible. Microarrays • • Microarrays are used to detect messenger RNAs from many genes simultaneously, to get a semiquantitative estimate of gene expression in a given cell type or growth condition. Probes from many different genes are placed in an array on a glass microscope slide, then hybridized to cDNA made from messenger RNA isolated from a tissue. You see which genes are active in that tissue. – Mostly done with 60mers: 60 bases long, synthetic oligonucleotides, made using sequence information from the genes. – cDNA is fluorescently labeled • • Often 2 conditions are compared (control and experimental), using red and green fluorescent tags. Semi-quantitative Determining DNA Sequence • We are going to discuss 2 DNA sequencing methods. – The Sanger sequencing method is currently thought to produce the most accurate and – • • longest sequences of any method. However, it is slow and expensive. It was invented in 1976, and was the only practical sequencing method for many years. Illumina sequencing is a currently popular type of Next-generation sequencing, a group of methods invented starting around 1995. These methods are massively parallel and generate huge amounts of data quickly and cheaply. The Sanger method uses DNA polymerase to synthesize a second DNA strand that is labeled. DNA polymerase always adds new bases to the 3’ end of a primer that is base-paired to the template DNA. An essential part of Sanger sequencing is chain terminator nucleotides: dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3' carbon of the deoxyribose. When DNA polymerase inserts one of these ddNTPs into the growing DNA chain, the chain terminates, as nothing can be added to its 3' end. Sequencing Reaction • The template DNA is usually single stranded DNA, which can be produced from plasmid cloning vectors that contain the origin of replication from a single stranded bacteriophage such as M13 or fd. The primer is complementary to the region in the vector adjacent to the multiple cloning site. • Sequencing is done by having 4 separate reactions, one for each DNA base. All 4 reactions contain the 4 normal dNTPs, but each reaction also contains one of the ddNTPs. In each reaction, DNA polymerase starts creating the second strand beginning at the primer. When DNA polymerase reaches a base for which some ddNTP is present, the chain will either: – terminate if a ddNTP is added, or: – continue if the corresponding dNTP is added. – which one happens is random, based on ratio of dNTP to ddNTP in the tube. However, all the second strands in, say, the A tube will end at some A base: you get a collection of DNAs that end at each of the A's in the region being sequenced. • • • • Electrophoresis • • • The newly synthesized DNA from the 4 reactions is then run (in separate lanes) on an electrophoresis gel. The DNA bands fall into a ladderlike sequence, spaced one base apart. The actual sequence can be read from the bottom of the gel up. Automated sequencers use 4 different fluorescent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the same lane of the gel. – Today’s sequencers use capillary electrophoresis instead of slab gels. – Radioactive nucleotides (32P) are used for non-automated sequencing. • Sequencing reactions usually produce about 500-1000 bp of good sequence. Next Generation Sequencing • Recently a number of faster and cheaper sequencing methods have been developed. – – • We are going to discuss the Illumina sequencing method, which is probably the most widely used at present. But, there are several other common methods that can be called “next generation”: Ion Torrent, 454, SOLiD, and more. Third generation sequencing: getting long sequences from single molecules, is getting started. Applications: – sequencing of whole bacterial genomes in a single run – sequencing genomes of individuals – metagenomics: sequencing DNA extracted from environmental samples. The large majority of microorganisms can’t be grown in the lab, and have only been detected by sequencing DNA from the environment. – Deep sequencing: looking for rare variants in a single amplified region, in tumors or viral infections – RNASeq: sequencing total cellular mRNA converted to cDNA. – ChIP-Seq: identifying the binding sites of transcription factor proteins by immunoprecipitation of the proteins attached to the DNA, then sequencing the DNA. Illumina Sequencing • • • Many sequencing methods have been invented, and it’s still a very active area of research. Most use the concept of sequencing by synthesis: starting with a primer, use DNA polymerase to add new bases are added one at a time, paying attention to which base is added. In the Illumina method, fluorescent tags attached to the 3’ OH group are used. – • • • • Each of the 4 nucleotides has a different colored tag. The fluorescent tags block the 3’-OH of the new nucleotide, and so the next base can only be added when the tag is removed. A cycle: add one new base, then read its color, then remove the fluorescent tag to give a free 3’ OH group. Repeat the cycle up to 200 times. End up with 200 bp of sequence information. More Sequencing • • • To get enough signal from the DNA molecule being sequenced, each DNA molecule needs to be amplified using PCR. For the Illumina method, this is done by attaching individual DNA molecules to a solid surface, then PCR-amplifying them in place, giving tiny spots with about a million identical copies. The DNA polymerase sequencing reactions are then monitored with a high resolution video camera. Sequence Assembly • • • • DNA is sequenced in very small fragments: 100-1000 bp. Compare this to the size of the human genome: 3,000,000,000 bp. In shotgun sequencing (the usual method), DNA is fragmented randomly. Enough data is collected so each base is read 10 times or more on average. In principle, assembling a sequence is just a matter of finding overlaps and combining them. In practice: – most genomes contain multiple copies of many sequences, – there are random mutations (either naturally occurring cell-to-cell variation or generated by PCR or cloning), – there are sequencing errors and misreadings, – sometimes the cloning vector itself is sequenced – sometimes miscellaneous junk DNA gets sequenced Sequence Assembly • The big problem with all current sequencing methods: you only get very short reads: 200 bp maximum for Illumina, up to 1000 bp for the older (slower, much more expensive) Sanger method, etc. – The human genome is 23 DNA molecules (chromosomes) that total 3 billion bp. Human chromosomes are 50-250 million base pairs long. – You need to assemble the tiny reads into much longer contigs (continuous sequences). With a perfectly sequenced genome, the final contigs would be identical to the DNA sequence of the chromosomes. • How reads are assembled into contigs: overlapping sequences. Assembly Problems • • Chromosomes, especially eukaryotic chromosomes, are filled with sequences that are repeated many times. If you have a read from a repeated sequence, how do you know which copy it is? – Some repeats are next to each other (tandem repeats) and some are scattered all over the genome (dispersed repeats). The main solution to this problem is to start with longer DNA template molecules and sequence both ends. You don’t know the sequence in between, but you do know how far apart the ends are. This often allows you to jump over repeated sequences. – It’s not perfect, and even now there are no human chromosomes sequenced to 100% accuracy. RNA Seq • • This is a new method, published in 2008. It is probably the method of choice today for analyzing RNA content. Also called whole transcriptome shotgun sequencing. Very simple: isolate messenger RNA, break it into 200-300 base fragments, reverse transcribe, then perform large scale sequencing using Illumina or other massively parallel sequencing technology. – RNA sequences then compared to genomic sequences to find which gene is expressed and also exon boundaries – Exon boundaries are a problem with very short reads: you might only have a few bases of overlap to one of the exons. • • As with all RNA methods, which RNAs are present depends on the tissue analyzed and external conditions like environmental stress or disease state. Get info on copy number over a much wider range than microarrays. Also detects SNPs. Reporter Genes • • • We often wish to know the location of a gene product, within an organism or within a cell. One well-used method is to make an antibody against your protein and treat tissues mounted for microscopy with it. Your antibody is then detected with a second antibody that is conjugated with a fluorescent tag or an enzyme with an easily visualized product. It is often easier to use a DNA based method: replace the protein-coding part of the gene with an easily detected reporter gene. – – • This allows you to separate the effects of the gene control sequences from the effects of the protein itself. The reporter gene can be fused with just the promoter region, or it can be fused to the protein itself, where it acts as a separate domain. Often done in conjunction with confocal microscopy: examining the same image with visible light and fluorescence. Some Common Reporter Genes • GFP: green fluorescent protein. A small protein isolated from jellyfish. It requires no substrates or co-factors. Several variants give different colors: red fluorescent protein, for example. – It still works when it is fused to other proteins: it acts as a separate protein domain. • GUS: beta-glucuronidase. Produces an insoluble dye with an artificial substrate. Works well with plants and fungi because they don’t have any naturally occurring beta-glucuronidase. • lacZ: E. coli beta-galactosidase. Used in conjunction with the artificial substrate Xgal, which produces an insoluble blue dye. • Luciferase: from fireflies. Requires a substrate, but doesn’t require any UV stimulation, which means no autofluorescence by other compounds in the organism. Silencing Genes with RNAi • • • • • RNA interference (RNAi) can silence a gene by cleaving its mRNA. This is called a knockdown, and usually decreases the specific mRNA by 50-90%. RNAi is accomplishing by introducing into the cell a short double stranded RNA that matches the mRNA sequence you wish to attack. The double stranded RNA is processed by the Dicer enzyme, which inserts one strand into the RISC complex. This RNA strand is now called the guide RNA. The RISC complex with its guide RNA hybridizes to the mRNA, and then acts as an endonuclease to cleave the mRNA. Getting the double stranded RNA into the cell can be done by various transformation methods, or it can be made from a plasmid introduced into the cell.