AGRO/ANSC/BIOL/GENE/HORT 305 Fall, 2017 Recombinant DNA Technology (Chpt 20, Genetics by Brooker) Lecture outline: (#14) - RECOMBINANT DNA TECHNOLOGY is the use of in vitro molecular techniques to isolate and manipulate fragments of DNA - In the early 1970s, researchers at Stanford University were able to construct chimeric molecules called recombinant DNA molecules. Shortly, thereafter, it became possible to introduce such molecules into living cells. This achievement ushered in the era of gene cloning. - Recombinant DNA technology and gene cloning have been fundamental to our understanding of gene structure and function. GENE CLONING - The term gene cloning refers to the phenomenon of isolating and making many copies of a gene Table 20.1 summarizes some of the more common uses of gene cloning - Cloning experiments usually involve two kinds of DNA molecules -Chromosomal DNA: Serves as the source of the DNA segment of interest. - Vector DNA; Serves as the carrier of the DNA segment that is to be cloned. - The cell that harbors the vector is called the host cell. When a vector is replicated inside a host cell, the DNA that it carries is also replicated. - The vectors commonly used in gene cloning were originally derived from two natural sources: o 1. Plasmids o 2. Viruses - Commercially available plasmids have selectable markers, typically, genes conferring antibiotic resistance to the host cell. Plasmids have an origin of replication that is recognized in specific host cells. - Viruses can infect living cells and propagate themselves by taking control of the host s metabolic machinery. When a gene is inserted into a viral genome, the gene is replicated every time the viral DNA is replicated. - Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments - The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes. - These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand. Figure 20.1 shows the action of a restriction endonuclease. - Restriction enzymes were discovered in the 1960s and 1970s by Werner Arber, Hamilton Smith and Daniel Nathans. (Nobel Prize in 1978) - Restriction enzymes are made naturally by many species of bacteria. They protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophage. - Currently, several hundred different restriction enzymes are available commercially Table 20.3 gives a few examples - Restriction enzymes bind to specific DNA sequences. 1
- These are typically palindromic; the sequence is identical when read in the opposite direction in the complementary strand For example, the EcoRI recognition sequence is 5 GAATTC 3 CTTAAG - Some restriction enzymes digest DNA into fragments with sticky ends. These DNA fragments will hydrogen bond to each other due to their complementary sequences. - Other restriction enzymes generate blunt ends For example, the enzyme NaeI (Refer to Table 20.3) THE STEPS IN GENE CLONING: The general strategy followed in a typical cloning experiment is outlined in Figure 20.2 - The vector carries two important genes. o amp R à Confers antibiotic resistance to the host cell o lacz à Encodes β-galactosidase - Provides a means by which bacteria that have picked up the cloned gene can be identified - All bacterial colonies growing on the plate had to have picked up the vector and its amp R gene - In the hybrid vector, the chromosomal DNA inserts into the lacz gene, thereby disrupting it. - By comparison, the recircularized vector has a functional lacz gene. - The growth media contains two relevant compounds: o IPTG (isopropyl-β-d-thiogalactopyranoside): A lactose analogue that can induce the lacz gene. o X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside): A colorless compound that is cleaved by β-galactosidase into a blue dye - The color of bacterial colonies will, therefore, depend on whether or not the β- galactosidase is functional: o If it is, the colonies will be blue o If not, the colonies will be white - In this experiment, bacterial colonies with recircularized vectors form blue colonies, while those with hybrid vectors form white colonies - The net result of gene cloning is to produce an enormous amount of copies of a gene - During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector - Amplification of the gene occurs in two ways: 1. The vector gets replicated by the host cell many times. This will generate a lot of copies per cell. 2. The bacterial cell divides approximately every 30 minutes. This will generate a population of many million overnight. - Recombinant DNA technology is not only used to clone genes. Sequences such as telomeres, centromeres and highly repetitive sequences can be cloned as well. cdna can be made from mrna via Reverse Transcriptase - To clone DNA, one can start with a sample of RNA - The enzyme reverse transcriptase is used 2
- Uses RNA as a template to make a complementary strand of DNA - DNA that is made from RNA is called complementary DNA (cdna) - It could be single- or double-stranded Synthesis of cdna is presented in Figure 20.3 - From a research perspective, an important advantage of cdna is that it lacks introns. This has two ramifications: - It allows researchers to focus their attention on the coding sequence of a gene - It allows the expression of the encoded protein. Especially, in cells that would not splice out the introns properly (e.g., a bacterial cell). POLYMERASE CHAIN REACTION: - Another way to copy DNA is a technique called polymerase chain reaction (PCR). It was developed by Kary Mullis in 1985. Was awarded the Nobel prize in 1993. - Unlike gene cloning, PCR can copy DNA without the aid of vectors and host cells. The PCR method is outlined in Figures 20.5, 20.6 - The starting material for PCR includes: o o o Template DNA: should contain the region that needs to be amplified Oligonucleotide primers; Complementary to sequences at the ends of the DNA fragment to be amplified. These are synthetic and about 15-20 nucleotides long. Deoxynucleoside triphosphates (dntps). Provide the precursors for DNA synthesis. o Taq polymerase - DNA polymerase isolated from the bacterium Thermus aquaticus. This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases - PCR is carried out in a thermocycler, which automates the timing of each cycle - All the ingredients are placed in one tube. - The experimenter sets the machine to operate within a defined temperature range and number of cycles. - The sequential process of denaturing-annealing-synthesis is then repeated for many cycles. A typical PCR run is likely to involve 20 to 30 cycles of replication. This takes a few hours to complete. - After 20 cycles, a DNA sample will increase 2 20 -fold (~ 1 million-fold). After 30 cycles, a DNA sample will increase 2 30 -fold (~ 1 billion-fold) - For a PCR reaction, a researcher must have prior knowledge about the sequence of the template DNA, in order, to construct the synthetic primers. - PCR is also used to detect and quantitate the amount of RNA in living cells. The method is called reverse transcriptase PCR (RT-PCR). RT-PCR is carried out in the following manner: (Figure 20.7) o RNA is isolated from a sample o It is mixed with reverse transcriptase and a primer that will anneal to the 3 end of the RNA of interest. 3
o This generates a single-stranded cdna which can be used as template DNA in conventional PCR. RT-PCR is extraordinarily sensitive. It can detect the expression of small amounts of RNA in a single cell. DETECTION OF GENES AND GENE PRODUCTS Molecular geneticists usually want to study particular genes within the chromosomes of living species. This presents a problem, because chromosomal DNA contains thousands of different genes. - The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes. There are several different techniques to choose from. DNA libraries - A DNA library is a collection of thousands of cloned fragments of DNA. o When the starting material is chromosomal DNA, the library is called a genomic library. o A cdna library contains hybrid vectors with cdna inserts The construction of DNA libraries is shown in Figure 20.11 Screening of libraries: - In most cloning experiments, the ultimate goal is to clone a specific gene. For example, suppose that a geneticist wishes to clone the rat β-globin gene. - Only a small percentage of the hybrid vectors in a DNA library would actually contain the gene. Therefore, geneticists must have a way to distinguish those rare colonies from all the others. - This can be accomplished by using a DNA probe in a procedure called colony hybridization. Refer to Figure 20.12 The DNA probe? - If the gene of interest has been already cloned, a piece of it can be used as the probe. - If not, one strategy is to use a probe that likely has a sequence similar to the gene of interest. For example, use the rat β-globin gene to probe for the β-globin gene from another rodent. - For a novel type of gene that no one else has ever cloned from any species. If the protein of interest has been previously isolated, amino acid sequences are obtained from it. These amino sequences can be used to design short DNA probes that can bind to the protein s coding sequence. Southern Blotting is used to detect DNA sequences - Southern blotting can detect the presence of a particular gene sequence within a mixture of many. It was developed by E. M. Southern in 1975. - Southern blotting has several uses: 1. It can determine copy number of a gene in a genome. 2. It can detect small gene deletions that cannot be detected by light microscopy 3. It can identify gene families. 4. It can identify homologous genes among different species Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene, should have been cloned. - This cloned DNA is labeled (e.g., radiolabeled) and used as a probe. The probe will be able to detect the gene of interest within a mixture of many DNA fragments 4
The technique of Southern Blotting is shown in Figure 20.15 - Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules - Northern blotting has several uses: 1. It can determine if a specific gene is transcribed in a particular cell type. Nerve vs. muscle cells. 2. It can determine if a specific gene is transcribed at a particular stage of development. Fetal vs. adult cells. 3. It can reveal if a pre-mrna is alternatively spliced Figure 20.16 shows the results of a Northern blot for mrna encoding a protein called tropomyosin - Smooth and striated muscles produce a larger amount of tropomyosin mrna than do brain cells. This is expected because tropomyosin plays a role in muscle contraction. - The three mrnas have different molecular weights. This indicates that the pre-mrna is alternatively spliced. - Western blotting is used to identify a specific protein within a mixture of many protein molecules Western blotting has several uses: - 1. It can determine if a specific protein is made in a particular cell type. Red blood cells vs. brain cells. - 2. It can determine if a specific protein is made at a particular stage of development. Fetal vs. adult cells Western blotting is carried out as such: - Proteins are extracted from the cell(s) and purified. - They are then separated by SDS-PAGE - They are first dissolved in the detergent sodium dodecyl sulfate - This denatures proteins and coats them with negative charges - The negatively charged proteins are then separated by polyacrylamide gel electrophoresis - They are then blotted onto nitrocellulose or nylon filters - The filters are placed into a solution containing a primary antibody (recognizes the protein of interest) - A secondary antibody, which recognizes the constant region of the primary antibody, is then added - The secondary antibody is also conjugated to alkaline phosphatase - The colorless dye XP is added - Alkaline phosphatase converts the dye to a black compound - Thus proteins of interest are indicated by dark bands Figure 20.17 shows the results of a Western blot for the β-globin polypeptide Techniques that Detect the Binding of Proteins to DNA - To study protein-dna interactions, the following two methods are used o 1. Gel retardation assay. Also termed band shift assay. o 2. DNA footprinting The technical basis for a gel retardation assay is this: 5
- The binding of a protein to a fragment of DNA retards its rate of movement through a gel. (Figure 20.18) - Gel retardation assays must be performed under nondenaturing conditions. - Buffer and gel should not cause the unfolding of the proteins nor the separation of the double helix DNA Sequencing: - During the 1970s two DNA sequencing methods were devised. One method, developed by Alan Maxam and Walter Gilbert, involves the base-specific cleavage of DNA. The other method, developed by Frederick Sanger, is known as dideoxy sequencing. - The dideoxy method has become the more popular and will therefore be discussed here. (Figure 20.12) - The dideoxy method is based on our knowledge of DNA replication with DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5 P of one and the 3 OH of the other Nucleotides missing that 3 OH can be synthesized - Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow. This is referred to as chain termination. - Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts. This is accomplished using cloning or PCR techniques. - In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site - If double-stranded DNA is used as the template, it must be denatured at the beginning of the experiment - An important innovation in the method of dideoxy sequencing is automated sequencing - It uses a single tube containing all four dideoxyribonucleotides - However, each type (dda, ddt, ddg, and ddc) has a different-colored fluorescent label attached. - After incubation and polymerization, the sample is loaded into a single lane of a gel. - The procedure is automated using a laser and fluorescent detector. - The fragments are separated by gel electrophoresis, the mixture of DNA fragments are electrophoresed off the end of the gel. - As each band comes off the bottom of the gel, the fluorescent dye is excited by the laser - The fluorescence emission is recorded by the fluorescence detector. The detector reads the level of fluorescence at four wavelengths 6