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BCH 401G Lecture 40/41 Andres Lecture Summary: DNA Technology Human Genome contains approximately 3 x 109 bp of DNA and is composed of approximately 100,000 different genes. We will talk about five technologies which have had a major impact on science this century: 1. Restriction Endonucleases. 2. Plasmid vectors. 3. DNA sequencing. 4. Polymerase Chain Reaction. 5. Complementary DNA (cDNA) Cloning. 1. Restriction Endonucleases Restriction Endonucleases break the 3',5' phosphodiester bonds between nucleotides. Different enzymes break this bond on different sides of the bond. Most restriction endonucleases recognize DNA palindromes. Restriction enzymes are used to cleave DNA molecules at defined points to yield specific fragments that are more readily analyzed and manipulated than the parental DNA molecule. DNA ends generated by digestion with restriction enzymes are called "sticky ends" or overhangs. These overhangs can base-pair with ends of DNA molecules cut with the same restriction endonuclease. This is a very useful feature: This activity can be used to rejoin fragments. Restriction Endonucleases are important because they allow large pieces of DNA to be cut into smaller defined fragments so we can isolate and study them. Most common method for analyzing DNA digestions is by agarose gel electrophoresis. Agarose is a material which can be used to separate DNA molecules on the basis of size. Mixture of DNA fragments is placed in a trough cut into one end of the agarose gel. A negative electrode is placed at the end of gel nearest the DNA and a positive electrode at other end of gel. Because of negatively charged phosphates in backbone of DNA, DNA moves toward positive electrode. II. Plasmid vectors. Restriction enzymes, DNA ligase and plasmid vectors are the cornerstone of recombinant DNA technologies. They allow the production of new combinations of unrelated genes to be constructed in the laboratory. These new genes can be CLONEDamplified many times-by placing them into a suitable host. Theses new genes are often transcribed and translated. These techniques are very powerful because once these engineered proteins are introduced to a host cell, the host cell can be permanently and profoundly altered. Cloning a segment of DNA entails five general procedures: 1. A method for cutting DNA at precise locations (rest. enzymes). 2. A method for joining two DNA fragments covalently (DNA ligase). 3. Selection of a small molecule of DNA capable of self-replication. Segments of DNA to be cloned can be joined to plasmids, These composite molecules are called recombinant DNAs. 4. A method for moving recombinant DNA from the test tube into a host cell that can provide the enzymatic machinery needed for DNA replication. 5. Methods to select or identify those host cells that contain recombinant DNA. The collection of techniques used to carry out these processes are collectively referred to as recombinant DNA technology or genetic engineering. Plasmid Cloning: 1. Plasmids (vectors) are small (2-6kb) circular DNA molecules found in bacteria. They are closed circular DNA duplex molecules and act as accessory chromosomes (see below). 2. They have Origins of Replication -can be replicated independently within bacteria. 3. Contain a selectable marker- most often provides resistance to an antibiotic. The drug is used to isolate only those bacteria which have received the plasmid DNA molecule. Plasmid vectors are useful - allow scientists to generate large amounts of specific DNA fragments for study. 3 Steps to using plasmid vectors 1. Cut target DNA (DNA to be propagated) and plasmid DNA with same restriction enzyme(s). Both DNAs now have same "sticky ends" 2. Mix cut target DNA and plasmid DNA together and add DNA ligase. Target DNA is inserted (ligated) into the plasmid. 3. Plasmid DNA with inserted target DNA sequence is then put back into bacteria. Called Transformation. Bacteria grow and divide rapidly- also replicate plasmid with inserted sequence. Bacteria can divide once every 30 minutes. This means that very soon you have a lot of bacteria and therefore a lot of plasmid DNA. You can then extract the plasmid carrying the DNA insert from the bacteria. 3. DNA Sequencing. How do you determine the sequence of DNA? The most common method for sequencing DNA is called the "Sanger Method". Frederick Sanger developed this method in 1976 and won his second Nobel Prize. This method depends on dideoxyribonucleotide triphosphates: ddNTPs There are four: ddATP, ddCTP, ddGTP, and ddTTP. ddNTPs have a hydrogen at the 3' position of the sugar instead of a hydroxyl group. Remember that the 3' hydroxyl group of the nucleotide is critical for the DNA synthesis reaction. Since dideoxyNTPs have no 3' hydroxyl group, the growing DNA chain can not be extended any further upon addition of a ddNTP. DNA synthesis terminates at that point in the DNA strand. Basic ingredients required for Sanger Sequencing method: 1. DNA you want to sequence and short single-stranded DNA primer (usually 17-25 nucleotides) which is complementary to the DNA to be sequenced. DNA to be sequenced is often prepared as single-stranded DNA. 2. Four dNTPs (dATP, dCTP, dGTP, and dTTP). 3. Four ddNTPs (ddATP, ddCTP, ddGTP, and ddTTP). 4. One dNTP which has a radioactive Alpha phosphate. 5. A DNA Polymerase -several different ones can be used. Method: 1. Mix primer-DNA complex with all four dNTPs. 2. Divide into four portions. To each portion add one (and only one) of the dideoxyNTPs. One gets ddATP, another gets ddCTP, and so on. Important: Every reaction contains all four deoxyNTPS and only one dideoxyNTP. Terminology: Reaction that contains ddATP is called "A" reaction, ddCTP is "C" reaction, etc. 3. Then add DNA polymerase enzyme. Polymerase extends primer and synthesizes DNA sequence by template DNA and incorporates one or more radioactive dNTPs. This allows you to detect the DNA that is being newly synthesized. ddNTPs are occasionally incorporated and act to terminate DNA synthesis. 4. Separate DNA fragments in each reaction by gel electrophoresis. DNA fragments separated by size. Length of the terminated fragments indicates the positions where the dideoxyNTP was incorporated in place of the deoxyNTP. Have "A" Lane (ddATP), "G"Lane (ddGTP), "C" Lane (ddCTP), and "T" Lane (ddTTP). Can then read DNA sequence from the gel. Important to understand: 1. There are very many template DNA molecules in each of these reactions. 2. Concentration of ddNTPs is such that they will only occasionally incorporate and terminate the synthesis. 3. Therefore, when each sequencing reaction is completed, there will be a variety of DNA products of different lengths. Can vary the amounts of dideoxyNTPs in the sequencing reaction. If you increase the amounts of dideoxy NTPs in the reaction the dideoxyNTPs will be more likely to be incorporated. Synthesis will terminate more often. You will be able to read the sequence close to the primer (near the start) on the sequencing gel. If you decrease the amounts of the dideoxyNTPs in the reaction, synthesis will proceed further because it will not terminate as often. This will allow you to read sequence further from the primer. 4. Another very powerful DNA Technology is the Polymerase Chain Reaction or PCR. PCR allows you to reproduce specific DNA sequences. The process is termed "Amplification". PCR generates large amounts of a particular DNA sequence from very small amounts of starting material (can achieve a billion fold amplification in a very short period of time). For PCR you need: 1. DNA which contains the sequence you want to amplify. 2. DNA Primers flanking the sequence you want to amplify. 3. dNTPs. 4. Heat-stable DNA polymerase. Three steps in PCR: 1. Denaturation. Heat to 95°C. Double stranded template DNA denatures (the double stranded DNA helix becomes two separate single stranded templates for PCR). 2. Annealing. Reaction is cooled to temperature below the Annealing temperature of the primer. Say 60°C. Primers now can base-pair with single-stranded DNA template. 3. Extension. Polymerase extends both primers and replicates DNA sequence (just a DNA polymerase reaction, need template, primer with a free 3'-OH, and dNTPs). Three steps called one cycle of PCR. Each cycle doubles the number of copies of the DNA sequence that lies between the primers. PCR can analyze small amounts of DNA. 40 cycles of PCR can allow you to detect 50 molecules of DNA (thirty cycles will produce about 1 million replica sequences from a single starting DNA sample). Since each cell has two copies of DNA, this means you can easily detect any DNA sequence starting with only 25 cells. Diagnostic and Forensic Medicine. Application of Restriction Mapping and PCR. Most of the DNA sequence in all humans is identical. However, there are differences between all of us that make us unique. Some of these differences create or remove Restriction Enzyme cleavage sites. This creates differences in sizes of fragments resulting from digestion of chromosomal DNA with restriction enzymes between individuals in any population. Called Restriction Fragment Length Polymorphism, or RFLP. This technique can be used to map the position of genes responsible for inherited human diseases. Many mutations either create or destroy a previously existing restriction enzyme site(s). If such a mutation occurs within or near a particular gene, the restriction enzyme pattern will change, and can be detected using radioactively labeled DNA probes. RFLP Analysis can be combined with PCR for Forensic Medicine. Examples: 1. Crime scene analysis: Can use PCR to produce a large amount of DNA from a small amount of initial material. RFLP analysis could then be used to compare digestion's of this DNA with a large number of known human markers to determine whether the DNA from a suspect is related to the crime scene sample. Must worry about contamination and analysis of a person from a small and isolated gene pool (an isolated group of individuals will tend to become inbred, this will tend to distort the representation of individual RFLP markers within this group relative to a larger population). 2. Test for disease, virus or microorganism. Specific DNA primers could be designed that would amplify the DNA from a foreign pathogen but not the human hosts DNA. 3. Medical Tests: If a specific gene mutation is correlated with a disease, PCR primers can be designed to detect the mutation. Infants or embryos (very early term) can then be tested for the potential of carrying a defective gene leading to illness. 5. Complementary DNA (cDNA) prepared from mRNA can be used to isolate active gene products: Remember that most Eukaryotic genes are mosaics of exons and introns. These interrupted genes can not be expressed in bacteria, however, this difficulty can be overcome by providing bacterial cells with recombinant DNA which is complementary to the mRNA. The key to forming complementary DNA (cDNA) is the enzyme reverse transcriptase. cDNA molecules can be inserted into plasmids that contain the necessary DNA regions for efficient expression in hosts such as bacteria, yeast, or mammalian cells grown in culture. In bacterial expression vectors, the cDNA is inserted into the vector in the correct reading frame near a strong bacterial promoter. In addition, to assure efficient translation a shine-dalgarno sequence is positioned near the AUG initiation codon. If cDNAs are made from the entire collection of mRNAs being expressed within a tissue or organism the collective cDNA clones when placed in a suitable plasmid are called a "cDNA library." This cDNA library should contain the entire repertoire of proteins being used by the host cells just before mRNA was collected. These "libraries" can then be used to isolate gene products of interest. This allows a researched to work in the opposite direction of the information flow we have studied. If a protein sequence is known, you could guess at the DNA sequence and using this "guess" look for the gene that encoded the protein you wished to study (think about how this could be done - it is often accomplished using PCR).