1 MITE 2 S Biology Biotechnology Summer 2004 Austin Che Biotechnology is difficult to define but in general it s the use of biological systems to solve problems. Recombinant DNA consists of DNA assembled from several pieces of DNA. Restriction Enzymes Restriction systems in bacteria destroy incoming foreign DNA. Viruses inject their DNA into bacteria in an attempt to take over the cell. In return, bacteria chop up the DNA into many pieces, restricting the entry of the virus. A restriction enzyme cuts DNA at a specific site. The recognition sequence for many restriction enzymes is an inverted repeat and usually 4, 6, or 8 bases long. Restriction enzymes can leave either blunt or sticky ends. Sticky ends are often more useful as the DNA will come together in a specific manner. The base pairing of matching sticky ends is temporary. DNA ligase is used to join DNA fragments together. Blunt end ligation is much less efficient than sticky end ligation. The names of restriction enzyme come from the initials of the bacteria they came from. EcoRI is a restriction enzyme from Escherichia coli strain RY13 and the I (one) indicates it was the first enzyme found in this strain. A modification enzyme recognizes the same site as a restriction enzyme and protects the DNA from being cut.
2 Biotechnology 2 Restriction fragment length polymorphism (RFLP) analysis looks at the length of fragments generated by cutting a piece of DNA with restriction enzymes and is used for DNA fingerprinting. Plasmids Vectors are molecules used to carry cloned genes. The most widely used vectors are bacterial plasmids. A plasmid is a circular molecule of DNA that can replicate independently of the bacterial chromosome. To select for plasmids, we incorporate an antibiotic resistance gene on the plasmid. Ampicillin, a member of the penicillin family, is widely used. The ampr gene confers ampicillin resistance. Plasmids are often engineered to have a multiple cloning site, which is a sequence of DNA with several restriction enzyme sites. PCR The polymerase chain reaction (PCR) is one of the most widely used method to amplify DNA and is brilliantly simple. For PCR, we need some DNA to serve as the template. In principle, a single template molecule can be amplified. For example, blood from a crime scene could be used as the template. We also need two PCR primers, two single stranded pieces of DNA that match the sequence at either end of the target sequence. Remember that DNA polymerase always requires a primer and cannot start a DNA chain by itself! We use a heat resistant DNA polymerase such as Taq polymerase from
3 Biotechnology 3 Thermus aquaticus (lives in the hot springs of Yellowstone). This polymerase can work (and prefers) high temperatures. We add nucleotides for the polymerase to make new DNA. We then take the mix and put it in a thermocycler to rapidly change the temperature. The basic PCR cycle consists of the following: ˆ Denaturation: Heat at 95 to denature the DNA, making them single stranded. ˆ Annealing: Heat at about 55 to allow the DNA to renature. The DNA will bind with the primers, which are in great excess of the template. ˆ Extension: Raise the temperature to the ideal working temperature of the polymerase, about 70, and allow the polymerase to extend all the primers. Every cycle of PCR can in theory double the amount of DNA, leading to exponential amplification. PCR is relatively fast usually taking a couple hours. In addition, the specific region of DNA between the primers rapidly outnumbers all other pieces of DNA. The longer the primers, the more specific the binding reaction. We don t always want to be specific! PCR is not only useful for amplification. We can also add arbitrary bases on the 5 end of the primers. As long as the 3 end matches the template, the polymerase will extend the DNA. This is often used to add restriction sites for cloning. PCR can also be used to deliberately introduce mutations into a gene.
4 Biotechnology 4 RT-PCR stands for reverse transcriptase-pcr and can be used to amplify from RNA. RNA is made into complementary DNA (cdna) and then amplified by PCR. cdna for eukaryotic genes are easier to clone as they do not contain introns. Electrophoresis Electrophoresis is the separation of charged molecules in an electrical field. In solution, a molecule moves in proportion to its charge/mass ratio. The charge/mass ratio of all DNA is the same! To separate by size, we use a gel, often made from agarose or acrylamide. A gel is like a mesh that slows down the molecules. In particular, larger molecules are slowed down more than small molecules. The distance traveled in the gel is inversely proportional to the logarithm of the molecular weight. The speed of migration can be controlled by the percentage of gel used. Ethidium bromide is a dye that stains DNA and RNA allowing us to visualize the bands on the gel. To find the length of an unknown piece of DNA, we run a standard ladder that contains known lengths of DNA. Proteins can be positive, negative, or usually uncharged. For proteins, sodium dodecyl sulfate (SDS), a detergent, is used to unfold protein. SDS contains a negative charge giving the protein a negative charge in proportion to its length like DNA. As proteins are usually much smaller than DNA/RNA, polyacrylamide with smaller gaps than agarose is used.
5 Biotechnology 5 Transformation Transformation is the process of inducing bacteria to take up DNA. There are several ways to make cells take up DNA, such as applying a high voltage to create holes in their membrane, called electroporation. Gene Expression E. coli is often used to overproduce a protein of interest. As high expression is desired, a strong promoter and RBS is necessary. However, high expressions are often lethal to the cell, so expression should also be inducible. Optimizing codon usage can also maximize expression. Expression can also be controlled by changing the plasmid s copy number. Some plasmids can be induced to go from being low copy to high copy. After growth in a host like E. coli, it is necessary to separate the molecule of interest from the other junk floating around. Lysozyme is an enzyme that digests bacterial cell walls and detergents dissolve the cell membrane. This breaks open cells releasing their contents. Ribonuclease is an enzyme that destroys RNA but not DNA. Proteases destroy proteins. DNAses destroy DNA. Reporter Genes Reporter genes are easy to detect and inserted for debugging purposes. Some examples include antibiotic resistance genes, such as for ampicillin, green fluorescent protein (GFP), lacz, or luciferase. To determine activity of an arbitrary gene, we can create a gene fusion, by attaching our gene to a reporter gene.
6 Biotechnology 6 DNA Sequencing The DNA sequencing method today uses a PCR like reaction. It is called the dideoxy method and invented by Fred Sanger. Because dideoxy nucleotides (ddntps) are missing an OH group at the 3 end, DNA chains with ddntps cannot be extended. Unlike PCR, only a single primer is inserted into the tube and a small amount of ddntps is inserted. The ddntps are inserted at random locations during extension leading to many different length products. We can have 4 different tubes to find locations of all four bases. The different length products are separated by gel electrophoresis and the sequence can be read directly. Today, sequencing is done in one reaction using all four ddntps together. 4 different fluorescent dyes are attached to each ddntp. There is a limit on the number of bases that can be sequenced per reaction (about 750). Primer walking involves sequencing a long piece of DNA by first sequencing one piece, making a primer that matches the end of the sequenced piece, and re-sequencing. Shotgun sequencing involves taking a large piece of DNA and randomly breaking it up into many small pieces and cloning it into a plasmid. The plasmid is grown up in E. coli and the DNA extracted. Primers that match DNA on the plasmid are used for sequencing. Reconstructing the original sequence involves piecing together many fragments from overlapping regions. The human genome is 3 billion base pairs and has been completely sequenced (or as completely as it ever will be with the current methods).
7 Biotechnology 7 Compare that with HIV with 9,800bp, E. coli with 4.6 million bp, Lilium longiflorum (trumpet lily) of 90 billion bp, and Amoeba dubia of 670 billion bp! Proteins can also be sequenced but is more difficult. Sanger also sequenced the first protein (insulin). Sanger received two Nobel prizes: one for DNA work and one for protein work. DNA Synthesis The currently used method for chemical synthesis of DNA involves phosphoramidite chemistry. The first nucleotide is anchored to a solid support like a bead. The nucleotides are added one at a time and washed at each step. To ensure only the correct group reacts at each step, many protecting groups are needed. Living systems have no need for protecting groups and are much faster! Each nucleotide addition takes about 5 minutes. Compare that to DNA polymerase in cells of 1000 nt/sec. Chemical synthesis proceeds in the 3 to 5 direction, opposite from all nucleic acid synthesis in living systems. Each nucleotide is not added with 100% so this method only works for short (about 100nt) oligonucleotides. Hybridization Hybridization utilizes the fact that a probe is complementary to a target sequence. For example, if we re looking for a sequence of DNA that has 5 -TAGCTGCA-3, we could design a DNA probe with the sequence 3 -ATCGACGT-5. Hybridization allows one to perform a search for a desired molecule.
8 Biotechnology 8 Finding a 1000 base stretch in human genome is like finding a needle in 2 ton haystack. How does chemistry do it so easily? As hybridization doesn t work well on a gel, the contents of a gel can be transferred to a nitrocellulose filter or nylon membrane. The filter can then be incubated with a probe to see where the probe binds on the filter. This process is called blotting. A Southern blot uses a DNA probe for DNA on the filter. A Northern blot probes with DNA or RNA for RNA on the filter. A Western blot probes using an antibody for protein on the filter. A Southwestern blot probes with DNA for protein on the filter. The probes can be radioactively or otherwise labeled. Biotechnology Applications New pathways can be constructed. Yeast, through fermentation, is able to convert glucose to alcohol. Cellulose is a polymer of glucose and yeast normally cannot break it down. Paper is almost entirely cellulose. Can we break down paper and turn it into alcohol? If in the US, we converted 100 million tons of waste paper/year into fuel alcohol, we could reduce gas usage by 15 percent. Cellulose can be broken down in four steps by 4 enzymes. These enzymes have been genetically engineered and cellulose can be broken down into glucose. Then yeast can convert it into alcohol. Indigo is used as a blue dye, e.g. blue jeans. E. coli have been engineered to produce indigo. Insulin is a protein hormone that controls blood sugar level. It is produced when blood glucose levels are high. Insulin was the first commercially available genetically engineered hormone for humans.