Recombinant DNA Technology Help (page 2)
Recombinant DNA Technology
Two primary advances helped usher in the new era of molecular genetics in the late 1970s to the early 1980s: (1) the use of recombinant DNA technology (genetic engineering) used to isolate and manipulate genes in vitro in order to endow cells with new synthetic capabilities and (2) the ability to synthesize and determine the linear order of nucleotides of DNA molecules (DNA sequencing). Recombinant DNA technology has made it possible to clone (isolate and make copies of individual genes) and transfer genes between bacterial species and strains or from eukaryotes into bacteria (or vice versa), causing the engineered cells to produce, sometimes in relatively large quantities, proteins of great economic importance such as enzymes (e. g. , amylases, proteases), hormones (e. g. , insulin, growth hormone), and interferons (lymphocyte proteins that prevent replication of many viruses). These proteins are made in such small quantities in human cells that the cost of their extraction and purification from blood or cadaver tissues has been very expensive, thus restricting their medical use in prevention and treatment of disease. Many products are now produced successfully using genetic engineering techniques; such products include blood-clotting factors (e. g. , tissue plasminogen activator, or TPA, used to activate the breakdown of blood clots and to prevent recurrence of blood clots in heart attack patients), and complement components (part of the immune system). In 1980, the United States Supreme Court decreed that new life forms created by genetic engineering could be patented, although this is not true in many other countries. This decision has contributed to the investment of large sums of money by private corporations into the development of many useful recombinant strategies. Public outcry against recombinant, or GM (genetically modified), crops in Europe had led the European Union (E. U. ) to ban or require extensive labeling requirements of foods that contain ingredients from GM crops. This has caused significant tension between the U. S. food industry and E. U. countries.
There are many hopes for the future benefit of genetic engineering technology in helping solve some of society's problems. In the agricultural arena, soybeans, corn, and cotton have been genetically engineered or modified to contain added herbicide tolerance and pest-resistance genes. These crops are also known asGMOs (genetically modified organisms). For example, one type of GM corn (Bt corn) contains a gene from the bacterium Bacillus thuringiensis that produces a protein that kills insect larvae. Larvae of the European corn borer can be costly pests for corn farmers. Having the pesticide gene in the corn eliminates the need to spray the crop with a pesticide. Other crops have been or are being engineered to contain genes that encode vaccines, added nutrients, and disease resistance, as well as resistance against cold, drought, and salinity. These few examples suffice to demonstrate the possibilities of this new technology and explain why there is such great excitement in the scientific, medical, agricultural, and pharmaceutical communities concerning its further development. While this technology is exciting and has proven useful, some have voiced concerns over potential environmental hazards, unknown risks to human health, and economic impact. These issues are still being investigated and must be resolved in order to make this technology useful and safe.
Most genetic engineering projects begin with the cloning, or isolation and amplification, of a gene. There are two major purposes for cloning a gene: (1) to obtain the sequence of its DNA (e. g. , in order to discover how an abnormal gene differs structurally from its normal allele) and (2) to produce large amounts of a gene product (i. e. , protein). Gene cloning generally involves three major steps: (1) A DNA fragment (or gene) of interest is isolated. (2) The fragment is spliced into a cloning vehicle called a cloning vector, such as a phage or a plasmid. (3) The vector is introduced into a host cell where it is replicated many times using the host cell's DNA replication machinery. A good cloning vector should have three characteristics: (1) the ability to replicate autonomously from the main chromosome to make many copies within the cell, (2) one or more unique recognition sites for restriction enzymes so it can be opened at a single position to receive a foreign DNA insert (i. e. , the gene of interest being cloned), and (3) a gene that encodes resistance to an antibiotic agent that will allow for the distinguishing of cells that contain the cloning vector as opposed to cells that do not.
Once the gene is cloned it can be analyzed (restriction mapped and its DNA sequence determined) and then transferred into a host cell or organism that will allow it to be expressed as a protein product. The host system might be a genetically engineered (transgenic) plant or a mammalian cell culture, yeast culture, or bacterial cell system that is used as a biological factory to produce large quantities of the desired product.
Cutting and Ligating DNA Molecules
- Enzymes that Create Cohesive Ends. DNA cut by restriction enzymes that leave complementary cohesive ends can be rejoined when mixed together in a test tube. They are brought together by their complementary sequences and then an enzyme called T4 DNA ligase seals the nicks. A common "cutting" and "pasting" scenario is shown in Fig. 12-1,where one of the cut fragments is a linear piece of genomic DNA from amammal and the other is a circular bacterial plasmid. When these two fragments come together in the presence of DNA ligase, a stable, covalently closed, circular recombinant DNA molecule is created. This recombinant molecule is a hybrid or chimera of DNA from bacteria and a mammal.
- Joining Blunt-Ended DNA Fragments. DNA fragments that contain complementary cohesive ends can be joined by the action of the enzyme DNA ligase, as discussed earlier. This enzyme, originally isolated from the bacterial phage T4, catalyzes the formation of a phosphodiester bond between adjacent nucleotides. This enzyme can also be used to join blunt-ended fragments together, but there are several methods that can be employed.
- Blunt-End Ligation. DNA ligase can be used to directly join the blunt ends of double-stranded DNA (ds DNA ) fragments. An advantage of this method is that it can join two defined sequences without introducing any additional material between them. The inability to control which pairs of blunt ends become joined is an obvious disadvantage (Fig. 12-2).
- Homopolymer Tails. An enzyme called terminal deoxynucleotidyl transferase can add any available deoxynucleotides to the 30 end of a single-stranded region of DNA without need of a template. If a string of A nucleotides are added onto the vector DNA and thymines (T) are added onto the foreign DNA , homopolymer tails are produced that should base-pair. The gap (absence of one or more nucleotides in a DNA molecule) can be filled by DNA polymerase I using the other complete strand as a template. The nick (absence of a phosphodiester bond between adjacent deoxyribose sugars) can be sealed byDNA ligase (Fig. 12-3).
- Linkers. A third method employs short DNA segments, called linkers or adapters, that contain a restriction enzyme recognition site; these can be synthesized chemically. Linkers can be added covalently to the ends of a plasmid or to an insert by blunt-end ligation (Fig. 12-4). This method imposes no restriction on the choice of sites to generate the ends, yet allows retrieval of the insert by cleavage with the appropriate restriction enzyme.
Blunt-ended DNA fragments can also be modified at their ends to become overhangs. These ends can pair with other complementary ends and be sealed by DNA ligase. There are two main methods for modification of blunt-ends.
It is also possible to convert cohesive ends into blunt ends by treatment with either an enzyme that will extend the shorter side of a 5' overhang or one that will degrade the cohesive end of a 3' overhang. Both of these methods employ the use of a DNA polymerase that either synthesizes new DNA in the case of extension or "filling in," or acts as a 3' to 5' nuclease in the case of degrading the 3' overhang. In either case, the original restriction enzyme recognition site is generally destroyed. In the following diagram, N-N represents any conventional complementary base pair.
Polymerase Chain Reaction (PCR)
Cloning DNA segments for use in a variety of purposes (i. e. , DNA sequencing) was formerly a relatively laborious in vivo process. However, in 1985, an in vitro technique, called the polymerase chain reaction (PCR), was developed for making large amounts of any DNA sequence without the need for cloning using gene banks or libraries. (see page 341). The PCR technique (Fig. 12-5) requires a pair of primers that are usually short (12–20 nucleotides long) pieces of chemically synthesized DNA (oligonucleotides), having nucleotide sequences specifically complementary to those on opposite strands flanking the target region to be copied.
These primers thus define the ends of the DNA segment that will be duplicated. The original template source of DNA does not have to be highly purified, and even a very small amount of template can serve as the initiator for the PCR. For example, cheek cells can be scraped from the inside of a cheek and placed directly into the PCR reaction. The DNA sample is heated, allowing the complementary DNA chains to separate (denaturation step). The primers are then added together with a heat-tolerant DNA -polymerizing enzyme. The primers bind to the single-stranded chains during the cooling phase (annealing step), and the polymerizing enzyme extends the primer through the rest of the fragment (extension step), creating double-stranded DNA molecules. The process is then repeated; i. e. , the mixture is reheated, and during the ensuing cooling phase the excess primers (or newly added primers) bind to template strands and become extended by the polymerizing enzyme to produce more double-stranded molecules. One cycle of denaturation, annealing, and extension can take several minutes, and about 20–30 cycles of heating plus DNA synthesis are normally run during gene amplification by the PCR (with heating and cooling times, this process can take several hours). After 20 cycles, a single DNA molecule can theoretically be amplified to about 1 million copies (called amplicons), and after 30 cycles to about 1 billion copies. With this quantity of DNA , nucleotide sequencing can be done easily.
Probes can also be used to locate genes or DNA segments of interest. Probes are commonly composed of a DNA sequence that is similar to the DNA or gene of interest. Because of the large quantity of DNA generated by the PCR, highly radioactive probes are not required, and the target segments can be detected by using nonradioactive probes or stains.
EXAMPLE 12. 4 Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome (AIDS). Very few susceptible cells actually harbor the virus in an infected person. It is estimated that 1–10 copies of viral DNA per million cells can be detected through the use of the PCR and suitable probes.
There are many variations of PCR. Multiplex PCR reactions contain primers for several different DNA targets, allowing for amplification of multiple, different sized DNA regions in a single PCR reaction. Nested PCR reactions consist of two successive amplification reactions, the first containing a primer set that targets a larger region of DNA. The second reaction contains primers that result in the amplification of a DNA target region within the first amplicon. This type of PCR approach helps reduce the amplification of non-specific DNA sequences.
A library is a collection of DNA fragments from a single source, such as a particular organism or a tissue. Libraries are created for the purpose of isolating a specific DNA fragment (or gene) of interest from a particular source. There are three general methods for achieving this by using recombinant DNA technology to (1) clone all of the genes of an organism into a genomic DNA library, (2) clone individual genes or DNA sequences of interest into a cDNA library, (3) clone gene fragments into an expression library, or (4) clone DNA fragments from an environmental sample into an environmental genomic DNA library. Identification of recombinant clones in a genomic or cDNA library can be carried out using a DNA probe that contains complementary sequences to the gene fragment of interest while identification of expression library recombinants requires the use of a protein specific antibody.
Genomic DNA Library. In shotgun experiments, genomic DNA from a donor organism is cut into many pieces by the same restriction endonuclease used to cleave the plasmid vector at its single restriction endonuclease site. The two kinds of fragments—various pieces of genomic DNA and the plasmid—are mixed in vitro and allowed to randomly rejoin by their complementary (cohesive, or sticky) ends to form circles (Fig. 12-1).
If it is important that a DNA insert be connected in the proper orientation into a vector for expression of the foreign DNA , the vector and insert can be cleaved with two restriction enzymes that generate different cohesive tails at the ends of each fragment. The insertion can then occur in only one orientation (Fig. 12-6).
Each new recombinant molecule is designed to contain a different piece of the genomic DNA. Recipient bacterial cells are made permeable for transformation by the naked DNA of the plasmids by treatment with a cold calcium chloride solution. When plated on nutrient agar containing an antibiotic, each transformed cell will multiply many times to form a colony or clone, all cells of which contain multiple copies (sometimes hundreds) of the same chimeric plasmid. The transformed cells can grow on the medium containing the antibiotic since they contain a gene that confers resistance to the drug. The large set of clones that collectively contains all the donor's DNA (i. e. , its genome) is known as a genomic DNA library. It should be noted that all the DNA is cloned, not just individual genes.
- Gene or cDNA Library. This method for isolating genes is much more specific than a genomic DNA library. Instead of cloning all of the donor's DNA , only specific genes (i. e. , open reading frames) or gene fragments are cloned. If the desired protein is very small (15–20 amino acids) or a short segment of protein sequence is known, it is possible (using the genetic code) to chemically synthesize a corresponding DNA molecule. One of the earliest protein-coding sequences synthesized in this manner was that for the hormone somatostatin (14 amino acids; 42 base pairs in the DNA strand).
- Expression Library. Expression libraries are built using vectors that contain very efficient regulatory sequences that allow genes to be highly expressed in host cells. These regulatory sequences are the promoters from host cell genes that are known to be efficiently expressed. Libraries of gene fragments can be created in expression vectors using the types of cloning procedures discussed for genomic libraries in section. These libraries are screened for clones able to produce the desired protein product. Alternatively, cloned genes can be fused to the strong promoters for expression of a useful gene product, such as insulin.
- Environmental genomic DNA Library. An environmental genomic library is created in the same way as a traditional genomic DNA library except that it contains DNA fragments from a population of organisms present in a sample taken from the environment, such as soil or ocean water. Instead of containing the genome of a single organism, it contains pieces of genomes from many different organisms, such as the hundreds of different types of bacteria that might be present in a soil sample. Environmental libraries can be screened for the presence of specific genes or used for sequencing the "genomes" present in the sample. This type of analysis is sometimes referred to as metagenomics because the organismal source material is unknown and of a community nature (i. e. , not from a single cultured source). It allows for the genetic study of organisms in their natural environment that may be impossible to isolate using standard culturing techniques.
EXAMPLE 12. 5 A portion of the protein sequence for somatostatin is shown below. The genetic code was used to perform reverse genetics (from protein to DNA ) in order to figure out a hypothetical DNA sequence for this protein. Because of the degeneracy of the genetic code, the third base position in many mRNA codons may possibly contain more than one nucleotide. The potential nucleotides that can fill this position are indicated as letters in the 3rd position of the codon and below this position. This synthetic piece of DNA is known as a degenerate oligonucleotide.
Larger synthetic genes have been built using (1) PCR or (2) a combination of oligonucleotide synthesis, PCR, and restriction enzyme cutting and ligation. Most proteins, however, are too long to allow chemical synthesis of the corresponding DNA. In this case, it is possible to isolate the total population of mRNA molecules from those cells that are specialized to make the protein. For example, human insulin is produced only by the pancreas, even though the insulin gene is present in all human nucleated body cells. A purified preparation of insulin mRNA can be isolated from pancreatic cells. A synthetic oligonucleotide of thymidines (oligo-dT) is hybridized to the poly-A tail of the mRNA strand. The viral enzyme reverse transcriptase (RNA-dependent DNA polymerase) is added to make a single-stranded DNA copy (cDNA or complementary DNA ) that ends in a hairpin loop (Fig. 12-7).
The mRNA template is then destroyed with alkali or an RNase enzyme. The hairpin end of the remaining cDNA serves as a primer for extension synthesis of a complementary strand byDNA polymerase I. The loop is then removed by an enzyme called S1 nuclease to produce a double-stranded cDNA molecule. These blunt-ended molecules can now be spliced into a suitable vector and cloned as previously described. Alternatively, linkers can be added to the ends of the cDNA to facilitate cohesive end cloning. A library of cDNA fragments is called a cDNA library. The advantage to this kind of library is that when cloning eukaryotic genes, DNA sequences that represent only the coding (exon) information will be obtained since mature mRNA does not contain introns.
A specialized type of cDNA library called an EST library (for Expressed Sequence Tag) is used to make a fairly quick assessment of the variety of expressed genes in an organism or tissue. This is done by sequencing hundreds of cDNA library clones and then comparing that sequence information with known databases of gene sequences.
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