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Recombinant DNA. OVERVIEW OF RECOMBINANT DNA TECHNOLOGY. Recombinant DNA technology allows a DNA fragment from any source to be joined in vitro with a nucleic acid vector that can replicate autonomously in microorganisms.
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OVERVIEW OF RECOMBINANT DNA TECHNOLOGY • Recombinant DNA technology allows a DNA fragment from any source to be joined in vitro with a nucleic acid vector that can replicate autonomously in microorganisms. • This provides a means of analyzing and altering genes and proteins. It provides the reagents necessary for genetic testing for carrier detection and prenatal diagnosis of genetic diseases and for gene therapy. • Additionally, this technology can provide a source of a specific protein, such as recombinant human insulin, in almost unlimited quantities.
Two approaches to producing recombinant DNA for cloning have been developed for use with somewhat different applications: • Cloning restriction fragments of cellular DNA • Cloning cDNA produced by reverse transcription of cellular mRNA
Cloning Restriction Fragments of Cellular DNA • Cloning DNA restriction fragments is useful in the following applications: • Sequencing DNA (Human Genome Project, Genetic diagnosis) • Producing restriction maps for gene mapping • Studies involving non-expressed DNA sequences
The first step in this procedure is to produce restriction fragments of DNA using restriction endonucleases. Restriction Endonucleases • These enzymes are isolated from bacteria, their natural source. • There are many different restriction endonucleases isolated from a variety of bacteria that are now readily available commercially. • In bacteria they act as part of a restriction/ modification system that protects the bacteria from infection by DNA viruses.
Restriction endonucleases recognize double-stranded DNA sequences called palindromes (inverted repeats) usually of four to eight base pairs in length. For example, Figure 1-6-1 shows the recognition site for EcoRI, a restriction endonuclease isolated from Escherichia coli. • A palindrome can be identified by examining the sequence of only one strand. Draw a line through the center of the sequence (through the central base for palindromes with an odd number of nucleotides). If the sequence is folded along this line the bases should pair.
Prokaryotic Restriction Modification Systems • Provide defense against infecting DNA viruses • Methylase enzyme modifies and protects palindromes in bacterial DNA. • Unmethylated palindromes of infecting viral DNA are recognized by restriction endonuclease. • Viral DNA is fragmented and destroyed.
DNA from a source to be cloned is mixed with a particular restriction endonuclease such as EcoRI, producing DNA restriction fragments. • Some restriction endonucleases such as EcoRI produce asymmetric cuts within the palindrome yielding "sticky ends" on the fragments. • Sticky ends are advantageous in facilitating the recombination of a restriction fragment with the vector DNA. • Others, like HaeIII, cut both strands in the same location yielding "blunt ends" on the restriction fragments.
Cloning Restriction Fragments Using Vectors • To clone the restriction fragments, they must each be inserted into a vector. • A vector is a piece of DNA (plasmid, viral chromosome, yeast chromosome) capable of autonomous replication in a host cell, for instance, plasmid pBR322. • The DNA used as a vector usually has. At least one type of palindrome recognized by a restriction endonuclease • An origin for autonomous replication • At least one gene for resistance to an antibiotic
Cloning vectors • Common properties • origin of DNA replication • unique restriction sites for insertion of DNA • multiple cloning sites containing many restriction sites engineered into many plasmid vectors • Easy identification and recovery of clones • Types of vectors • plasmids containing drug resistance gene (up to 10 kb) • many commercially available plasmids • bacteriophage, e.g., lambda (~ 15-20 kb) • cosmids for larger DNA molecules (~ 40-50 kb) • BAC: bacterial artificial chromosome (~ 100-300 kb) • YAC: yeast artificial chromosome (~ 1000 kb)
The vector is cut with the restriction endonuclease and mixed with the DNA restriction fragments to be cloned. As shown in Figure 1-6-3, once the vectors have combined with one of the restriction fragments, DNA ligase is used to form permanent PDE bonds between the fragment and the vector. This produces recombinant DNA.
Once the recombinant vectors have been produced, they are used to transform host cells. • In the example of the plasmid pBR322, the host cells are bacteria. Once transformed, the bacteria are plated on selective media so that bacteria transformed with a recombinant plasmid can be easily identified. • In the case of plasmid pBR322, bacteria with recombinant plasmids would be resistant to ampicillin but sensitive to tetracycline.
The collection of colonies produced is referred to as a genomic DNA library. The library must be screened with a radioactive probe to identify the colony with the desired restriction fragment.
Uses of Genomic Libraries • Large quantities of each clone can be grown for DNA sequencing studies, similar to what is being done in the Human Genome Project. • By producing genomic libraries using different restriction endonucleases (or allowing one type of restriction endonuclease to digest a DNA sample for different times), regions of overlap can be identified and the fragments ordered, producing DNA restriction maps useful for genetic testing and sequencing. • Other genetic markers may be identified in this way, such as minisatellite and microsatellite sequences. • Genomic libraries are also useful to clone and study DNA sequences that are not expressed in cells (response elements, introns, promoters).
Restriction Maps • A restriction map is a linear (or circular) map of the order and distances of restriction endonuclease cut sites in a segment of DNA • Each DNA fragment, no matter what size, has its own unique restriction map • Restriction maps are useful in comparing DNA fragments to look for regions of identity
Restriction Maps • Line drawings of DNA identifying sites cut by restriction endonucleases. • Identify potential RFLP markers for genetic diagnosis. • Example: Restriction site polymorphism for Mstll may be used to identify individuals with the sickle cell mutation
Cloning cDNA Produced by Reverse Transcription of Cellular mRNA • If the end goal of cloning is to have a cloned gene expressed in a cell, the entire coding sequence must be cloned intact. Furthermore, if a cloned eukaryotic gene is to be expressed in bacteria (to make recombinant proteins), the gene must not contain introns, which could not be processed in a prokaryotic cell. In these cases it is more convenient to clone cDNA rather than DNA restriction fragments.
Producing cDNA by Reverse Transcription of mRNA • Cytoplasmic mRNA is isolated from a cell known to express the desired gene. Reverse transcriptase, along with other components (Figure 1-6-4), is used in vitro to produce double stranded cDNA that is subsequently recombined with a chosen vector to produce the recombinant DNA for cloning. In this approach: • All genes expressed will be cloned along with the desired gene. • None of the non-expressed DNA in the cell will be cloned. • Each cDNA represents the complete coding sequence of a gene. • The cDNAs have no introns. • An expression library is produced at the end of the cloning procedure.
Expression Vectors • If the goal of the cloning procedure is to obtain a recombinant protein, appropriate sequences required for transcription and translation in the cloning host cell must be provided because they will not be part of the cDNA. • For instance, to produce recombinant human insulin in bacteria, a bacterial promoter and a Shine-Dalgarno sequence must be included in the cloning plasmid near the insertion site for the cDNA. • Figure 1-6-5 shows an example of an expression vector, pUC. In some expression vectors, other regulatory sequences such as operators are added to allow expression of the cloned gene to be controlled.
Uses of cDNA (Expression) Libraries • Once the recombinant expression vectors containing the cDNA inserts are produced, they are used to transform bacteria (or other host cells) and produce cDNA (expression) libraries. Expression libraries may be used to produce recombinant proteins that in some cases have significant advantages over isolating them from natural sources: • Larger quantities may be produced. Recombinant human insulin used to treat diabetics. • Natural source may carry risk of infection. Recombinant Factor VIII used to treat hemophilia A has helped reduce the incidence of HIV infection in hemophiliacs. Recombinant HbsAg is now used to immunize against hepatitis B, eliminating the risk of introducing a viral infection during vaccination.
Genes cloned as cDNA are used for gene therapy protocols and for producing transgenic animals. • cDNA probes, for many of the blotting techniques, are produced by cloning.
Screening libraries for a Specific DNA Sequence • Figure 1-6-6 shows how libraries are screened to identify a desired DNA sequence. The top circle represents either a genomic library or an expression library on a growth plate. • A blot is made from the plate. • Colonies on the blot are lysed and treated with a radioactive probe specific for the DNA sequence 32P-DNA) or recombinant protein (125I-antibody reactive with the recombinant protein).
An autoradiogram of the probed blot is produced and the radioactive colony identified. • Once the corresponding colony has been identified, a sample can be used to inoculate a large broth culture from which one can isolate the cloned DNA or the recombinant protein.
Common Sources of 32P-DNA probes • cDNA can be used as a probe to locate a genomic clone • A cloned gene from another species can be used to locate the human homolog. • An approximate DNA sequence can be deduced and synthesized in the laboratory provided the amino acid sequence of the protein is known.
Gene Therapy and Transgenic Animals • Gene therapy now offers potential cures for individuals with inherited diseases. The initial goal is to introduce a normal copy of the gene that is defective into the tissues that give rise to the pathology of the genetic disease. For instance, about 50% of the children with severe combined immunodeficiency have a mutation in the gene encoding the "γ chain common to several of the interleukin receptors. cDNA from a normal "γ-chain gene was used to transduce autologous cells from infants with X-linked SCID with subsequent correction of the defects in their T-cells and natural killer cells. • Gene transfer requires a delivery vector (retrovirus; adenovirus, liposome). • Only tissues giving rise to the disease pathology are targeted for gene therapy. • Normal gene is not inherited by offspring.
Transgenic Animals • Transgenic animals are produced by transferring cDNA into the pronucleus of a fertilized ovum. The resultant transgenic animal has the new gene (transgene) in all of its cells including its reproductive tissues. Transgenic animals are now widely used as experimental models in which to study human diseases. • A variation of this technique produces a knockout animal, in which a normal gene has been functionally eliminated. This may be done by site-specific mutagenesis. • Transgenic animals have a new gene (transgene) introduced into their germline. • All cells of a transgenic animal contain the transgene. • Transgene is inherited by offspring. • Knockout animals have a normal gene intentionally inactivated/destroyed. • Transgenic and knockout animals are used as models of human disease.
Gene Therapy • When using gene therapy to correct genetic deficiencies in humans, the cloned normal gene is targeted only to the tissues giving rise to the major symptoms. • For instance about 50% of the cases of severe combined immunodeficiency (SCID) are caused by mutations in the gene for a subunit common to several interleukin receptors. • The approach to gene therapy has been to introduce a normal cloned gene into the patient's bone marrow cells that will subsequently divide and differentiate to produce T and B lymphocytes expressing the gene. • These patients subsequently have improved immune function. Because the cloned normal gene has not been introduced into reproductive tissues any children of the patient would inherit the defective allele.
Review Questions Select the ONE best answer. 1. If a cystic fibrosis patient were to be treated by gene therapy, which type of cells should be targeted as host cells? A. Germ cells B. Epithelial cells C. T cells D. Hemopoietic stem cells
2. A pharmaceutical firm is interested in the bacterial production of thymidylate synthase in large quantities for drug-targeting studies. An important step in the overall cloning strategy involves the ligation of synthase cDNA into a plasmid vector containing a replication origin, an antibiotic resistance gene, and a promoter sequence. Which additional nucleotide sequence should be included in this vector to ensure optimal production of the thymidylate synthase? A. Operator sequence B. PolyA sequence C. Shine-Dalgarno sequence D. Attenuator sequence E. 3' -splice acceptor sequence
3. Restriction fragment length polymorphisms may be produced by mutations in the sites for restriction endonucleases. For instance, a single base change in the site for the nuclease SalI produces the sequence GTGGAC, which can no longer be recognized by the enzyme. The original sequence recognized by SalI was: A. GTAGAC B. GCGGAC C. CTGGAC D. GTCGAC E. GTGTAC
4. Scientists studying a common mutation in the LDL receptor gene have inserted the defective gene into fertilized murine ova. The altered ova are implanted in a foster mother and the progeny are used to study the effects of the mutant allele. The mice produced in this procedure would be referred to as: A. Knockout mice B. Transgenic mice C. Allogenic mice D. Cloned mice E. Somatic-cell engineered mice
5. A linear DNA fragment is cleaved with the individual restriction enzymes HindIII and SmaI and then with a combination of the two enzymes. The fragments obtained are: HindIII 2.5 kb; 5.0 kb SmaI 2.0 kb; 5.5 kb HindIII + SmaI 2.5 kb; 3.0 kb; 2.0 kb - Draw the restriction map. - The mixture of fragments produced by the combined enzymes is cleaved with EcoRI, resulting in the loss of the 3.0 kb band and the appearance of a band at 1.5 kb. Mark the EcoRI restriction site on the map.
