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Recombinant DNA Technology Isolating DNA • Chemically, DNA is a very simple compound, with little variation • between species. It is also chemically quite different from other macromolecules (proteins, carbohydrates, lipids). The basic steps: 1. 2. 3. • Break the cells open Remove proteins and other macromolecules Concentrate the DNA by precipitating it and re-suspending it in fresh buffer. Methods for breaking the cells open vary between tissues, but usually involve mechanical disruption in a buffer that inhibits DNAases. – – – Buccal swabs (cells from inside the cheek) are usually extracted by simply incubating them in a buffer containing detergent (to disrupt cell membranes) and a powerful protease (to destroy DNAase enzymes) Mechanical homogenizers (including kitchen blenders) work well for fibrous tissues. Some homogenizers are very small, to extract DNA from a hair follicle, for example. Some tissues can be frozen in liquid nitrogen and ground up mechanically DNA Separation • Separating DNA from other cell components relies on the fact that DNA is chemically different from proteins and other macromolecules. – – • • 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/chloroform extraction) Phenol is very nasty, and many methods have been invented to get around this step. DNA can be precipitated and then resuspended in a smaller volume using a high salt concentration plus ethanol (ethanol precipitation). DNA is usually 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. Transfection • How to get DNA back into the cell after manipulation in vitro. – Transfection means the cell takes up naked DNA from the environment and incorporates it into its genome. – In non-mammalian cells, this process is called transformation. “Transfection” is used for mammals because transformation also means going from normal to cancerous. • Need to get the DNA through the cell membrane and into the nucleus. • Transfection can be transient or stable. – Transient transfection produced DNA that is NOT incorporated into the genome. After 2-3 days it gets degraded and lost. – Stable transfection causes the transfected DNA to be incorporated into the genome, where it remains permanently. In most cases, the transfected DNA is incorporated into a random chromosomal location. More Transfection • Several transient transfection methods are available: – Lipofection: coating the DNA in lipid vesicles that fuse with the cell membrane. This is very efficient and common with mammalian cells. – Electroporation: if you subject a cell to a high voltage electrical field (say 20,000 volts per centimeter), temporary 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. • Stable transfection with viral vectors. The virus carries the DNA into the cell more efficiently than any other method. – You need to select the rare cells that have incorporated the DNA using a drug resistance gene as part of the transformation vector. The only cells that survive treatment with the drug have incorporated the foreign DNA. – Safety can be a problem: these vectors are derived from pathogenic viruses and some generate strong immune responses. – Also, random insertion of DNA into the genome can lead to mutations, including the induction of cancer. Viral Transfection • The process: – Genetic engineering, done using E. coli. This results in your engineered DNA (the transgene) inserted into a plasmid vector – Plasmid is transfected into a special cell line (packaging cell line) to add the viral coat. – The viruses produced by the packaging line can be used to transfect other cell lines. 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 • • One of the easiest ways to characterize DNA is to determine the positions of restriction sites: sequences that are cut by restriction enzymes (more formally, restriction endonucleases). Restriction enzymes are part of the defense systems used by bacteria against foreign DNA. – Foreign DNA entering the cell is cut by the Res, but host DNA is modified so it can’t be cut. • • • Restriction enzymes cut at 4-8 bp sequences that are usually inverted repeats: GTCGAC, for example. Hundreds of different REs, cutting at different sites, are available. Restriction sites are in fixed positions on the DNA. Digestion with single enzymes are with pairs of enzymes gives bands of fixed size on electrophoresis gels. These sizes can be put together to make a map of a DNA molecule. Needles in Haystacks • The primary purpose of molecular techniques in human genetics is to find and characterize genes responsible for genetic disease. • 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. DNA Polymerase • DNA polymerase is the enzyme that replicates DNA. To do this, it needs: – Single stranded DNA template molecule – Primer: a short piece of DNA or RNA base-paired with a region of the template – dNTPs: the 4 deoxy nucleoside triphosphates dATP, dCTP, dGTP and dTTP, which are the raw materials for the new DNA strand. – DNA polymerase attaches new nucleotides to the 3’ end of the primer, using the template strand as a guide to picking the proper nucleotide to add. Polymerase Chain Reaction • Based on DNA polymerase, the enzyme that replicates 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. – Starting at each primer, DNA polymerase adds new bases to the 3' ends to create the new second strand. – PCR is a cyclical process. Each cycle doubles the number of DNA molecules between the primers: exponential growth. – 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. SSR Genetic Markers • • • • • • . Microsatellites (Simple Sequence Repeats: SSRs. They are short (2-5 bases) sequences that are repeated several times in tandem: TGTGTGTGTGTG is 6 tandem repeats of TG. 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. Mapping SSRs is a matter of having PCR primers that flank the repeat region, then examining the PCR products on an electrophoresis gel and counting the number of repeats. SSRs are co-dominant markers: both alleles can be detected in a heterozygote. Allele-Specific PCR • For base change mutations (single nucleotide polymorphisms). • Use a primer whose 3’ base matches the mutation. Will amplify one allele but not the other because the 3’ end is not paired with the template in the wrong allele. 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. Most of them leave sticky ends: short single stranded regions that will hybridize with complementary ends. • 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. Enzyme that 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). 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 beta-galactosidase 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 using heat plus calcium chloride. •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: 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 Bacterial Artificial Chromosomes • • • • • BACs are the most common vector for large inserts such as eukaryotic genome projects. Based on the E. coli 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. Expression Vectors • Various types: – RNA only: use a vector that has a phage T7 promoter in front of the cloning site, and an inducible T& 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 baceria 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). Sources of DNA to Clone • Genomic DNA: cut up whole genome and clone small pieces. Advantage is, you get everything. Disadvantage is, a lot of it is junk. – Two general methods: • 1. randomly shear DNA into small pieces, then ligate linkers to the ends: oligonucleotides that contain a useful restriction site. • 2. partially digest the DNA with a restriction enzyme that has a 4 base recognition site. These sites will appear at random every 256 (44) base pairs. Take long pieces. • cDNA: DNA copy of mRNA, made with reverse transcriptase. Advantage: you just get the expressed genes. Disadvantages: you don't get control sequences or introns, and frequency depends on level of expression. • Synthetic DNA: synthesized de novo (for example multiple cloning sites or linkers), or made by PCR cDNA Synthesis •use oligo-dT primer, which binds to poly-A tail. •make the first DNA strand from the RNA using reverse transcriptase 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 eh loop with S1 nuclease (which cuts at unpaired bases) •Attach synthetic linkers with DNA ligase and clone into a vector. Hybridization • The idea is that if DNA is denatured (made single stranded, also called 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 clones – microarrays 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 say 60oC. Complementary DNAs find each other and stick. Need to wash off non-specific 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 • • • • • • The Southern blot is 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. Restriction Fragment Length Polymorphisms • • • • • RFLPs: the first DNA-based genetic mapping technique. Advantage: every individual has many variations in their DNA, so you don’t need a special set of marker mutations. Also, the markers are co-dominant so you can accurately determine the genotype. Probe is a fragment of a cloned gene (labeled). Genomic DNA is cut with a restriction enzyme. Polymorphic sites: the restriction site is present in some individuals but not in others (due to mutation). But, even if one site is missing, there will be another restriction site a little further away (a restriction enzyme with a 6 base site cuts on the average every 46 = 4096 bp). Then do a Southern blot and autoradiography. 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 • • • • A microarray is a set of short (20-60 bases) oligonucleotides bound to a glass slide. The microarray is hybridized with fluorescently labeled DNA. For gene expression analysis, messenger RNA isolated from a tissue, then converted to cDNA. You see which genes are active in that tissue. Often 2 conditions are compared (control and experimental), using red and green fluorescent tags. Semi-quantitative Tiling Arrays • Tiling arrays: Microarray chips with short probes that cover the entire genome (sometimes overlapping, sometimes with gaps between) – Transcriptome mapping. Use RNA converted to cDNA and labeled to detect transcribed regions, even if the RNA is very short or not polyadenylated. A surprising number of transcribed regions do not look like genes: short exons, RNA only genes. – Finding protein-binding regions: ChIP-chip (chromatin immunoprecipitation chip). Isolate DNA with proteins bound (histones, transcription factors, etc.) Then break up the DNA into small fragments by sonication, immunoprecipitate the protein of interest with bound DNA, remove the proteins and hybridize the DNA with the tiling array chip. Transcriptome Mapping Chromatin Immunoprecipitation chip SNP Detection • The problem with detecting single nucleotide polymorphisms (SNPs) is that you need to get good hybridization with a perfect match, and little or no hybridization with a 1 base pair mismatch. It is hard to do this for many different sequences simultaneously. • A simple solution: for each SNP location, have oligos on the chip for all 4 possible bases. The one that hybridizes best should be the correct one. – Remembering that many people are heterozygotes, so hybridizing to 2 alleles is common • There are many other applications for microarrays. DNA Sequencing Determining DNA Sequence • Originally 2 methods were invented around 1976, but only one is widely used: invented by Fred Sanger. – Sanger sequencing is currently thought to produce the most accurate and longest sequences of any method. However, it is slow and expensive. • 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. • Also uses 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. Sanger Sequencing Protocol 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 individual people or tumors metagenomics: sequencing DNA extracted from environmental samples Deep sequencing: looking for rare variants in a single amplified region, in tumors or viral infections – RNASeq: sequencing total cellular mRNA converted to cDNA. 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 (current favorite) , 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 454, 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.