7: Recombinant DNA

I. Introduction:

In the past fifteen years, we have witnessed a complete revolution in molecular genetics and biochemistry. This revolution has not been the result of a single new development in instrumentation or a theoretical breakthrough, but rather the application of a variety of techniques collectively referred to as genetic engineering or recombinant DNA technology. This new technology involves the in vitro modification and recombination of genetic material from different organisms to create new gene combinations; the product is often described as recombinant DNA. These techniques are applicable both to prokaryotes and eukaryotes.

Understanding the structure, function, and regulation of genes and their products is essential to an appreciation of biological systems. This also involves understanding the organization of an organism’s genome. Before the advent of recombinant DNA techniques, the understanding of gene regulation was hampered by the complexity of the average eukaryotic genome, which may contain up to 109 bp in approximately 50,000 genes. Investigators need to be able to isolate and study a gene in a purified form to analyze its structure and function. Molecular cloning of DNA provides a mechanism for isolating a single discrete segment of DNA from a population of genes, purifying this segment to homogeneity, and amplifying the DNA segment to produce enough pure material for chemical, genetic, and biological analysis.

A typical cloning experiment requires: (1) the DNA of interest (target DNA); (2) a cloning vector; (3) restriction endonucleases; (4) DNA ligase; and (5) a prokaryotic cell to serve as the biological host.

1. Target DNA: Almost any gene or DNA sequence can be cloned. The purpose for doing so range from basic research on gene structure, function, and regulation; to industrial applications such as over-expression of a biological molecule. DNA can be isolated from virtually any source: plant, animals, bacteria, fungi, etc.

2. Cloning Vectors: Bacterial plasmids are small (3-15 kb), closed circular, extrachromasomal pieces of DNA that replicate autonomously and can easily be placed into and extracted from their bacterial host. Since their initial discovery, certain plasmids have been highly modified in order to make them ideal for molecular cloning purposes. Unnecessary sequences have been deleted, polylinkers or multiple cloning sites have been inserted, their origin of replication has been modified to allow them to replicate to extremely high numbers within the bacteria cell (>400 copies/cell in some cases), and easily identifiable genetic markers (such as antibiotic resistance or the LacZ gene) have been inserted making identification of bacterial colonies containing recombinant plasmids easy and rapid. There are a large number of plasmids (referred to often as cloning vectors) commercially available for virtually any purpose.

3. Restriction Endonucleases: Restriction enzymes or restriction endonucleases cut (or digest) DNA in a site-specific manner. They were originally identified in bacteria where they protect the cell from infection by bacteriophages by destroying any DNA it recognizes as foreign (based on differences in methylation). Each type of restriction enzyme cleaves at its own unique sequence which is almost always a palindromic sequence from four to six base pairs in length. Some enzymes cleave the DNA in the exact center of their recognition sequence leaving even or blunt ends on the cut DNA but most enzymes cleave off-center and leave complementary single-stranded ends on the pieces of DNA. These are often referred to as “sticky ends” and are easier to ligate together than blunt-ended DNA. A large number of restriction enzymes (>150) are commercially available in purified form.

4. DNA Ligase: This enzyme catalyzes the formation of phosphodiester bonds between adjacent nucleotides (assuming one nucleotide has a free 3 hydroxyl group, and one has a free 5 phosphate). This means it can join two pieces of DNA together as one. DNA isolated from different sources that have been digested with a restriction endonuclease(s) can be ligated together provided they have complementary sticky ends or both have blunt ends.

5. Biological Host: Once the sequence of interest has been cut with restriction enzyme(s) and ligated into a cloning vector (which was digested with a compatible enzyme(s) it is placed into an Escherichia coli host strain for propagation. One method for introducing foreign genetic material (recombinant DNA molecules) into bacterial cells is called transformation. There are a number of E. coli strains that can be used for this purpose. Almost all contain DNA repair, methylation, restriction, and recombination deficient genotypes. This limits the ability of the bacteria to reject or mutate the foreign DNA. Once the bacteria are transformed, they are spread onto an agar plate. Each individual bacterium multiplies on the agar plate to form a colony. All the bacteria within a colony are genetically identical and are referred to as clones.

Once the recombinant molecules have been introduced into bacteria they must be screened to make sure they carry the correct recombinant molecule. The three most common methods of screening clones are antibiotic selection, insertional inactivation, and restriction enzyme digestion/agarose gel electrophoresis. Antibiotic selection requires a cloning vector caring an antibiotic resistance gene. When bacteria are plated on an agar plate containing the appropriate antibiotic, only those bacteria that are transformed will form colonies. This method of selection is almost performed immediately after transformation. Insertional inactivation requires a plasmid caring an additional antibiotic resistance or the LacZ gene. When the sequence of interest is cloned into the plasmid poly-cloning site, it interrupts this additional gene (either LacZ or antibiotic resistance). This causes inactivation of the gene. In the case of LacZ, bacteria containing non-recombinant plasmids (functional LacZ) will turn blue on an agar plate containing the chromogenic substrate X-gal. If LacZ is interrupted by the insertion of foreign DNA (recombinant molecule), the colonies will be unable to metabolize X-gal and will remain white in color. Using these two methods (antibiotic selection and insertional inactivation), the very first agar plate can separate transformed from non-transformed bacteria and distinguish bacteria colonies containing recombinant from bacteria clones containing non-recombinant DNA molecules. Once appropriate clones are identified, they are grown in larger quantity and their plasmid DNA is extracted. The plasmid DNA (or plasmid mini-prep) is then digested with restriction enzymes and the DNA fragments are resolved on an agarose gel. If the recombinant molecule is correct, the pattern and the size of the DNA fragments should be as predicted.

One recently developed technique which bypasses many of the traditional lengthy or cumbersome protocols involved in isolating the target DNA is the polymerase chain reaction, commonly referred to as PCR. This new technique was devised by Kary Mullis of Cetus Corp. in the mid 1980’s, and has since become common place in many laboratories. Using this method, a single target DNA sequence in any region of a genome (fly, mouse, human, etc.) can be rapidly amplified to thousands or millions of copies, thereby rendering it plentiful enough for sequencing or cloning. In brief, the PCR technique involves first the separation (denaturation) of the two strands of a double helical DNA by heating the DNA to a high temperature. Each of the two separated stands is then annealed at a lower temperature with one or two specific primers designed to hybridize to a sequence flanking the target region to be amplified. Polymerization reaction then extend inward from the two primers to create two new double stranded DNA molecules, each containing one newly-synthesized strand and one template strand. These reactions are catalyzed by a thermostable DNA polymerase (Taq polymerase). The DNA molecules are subsequently denatured again, hybridized again with the two flanking primers, and extended again by the Taq polymerase. This cycle of strand separation, primer annealing and polymerization can be carried out for many cycles, thereby exponentially amplifying the target DNA template. For instance, a unique sequence run through 30 cycles of PCR will be amplified 109 times (2Xn; where X=number of double stranded templates before cycle 1, and n=number of cycle).

II. Experimental Design:

In this section, we are going to experience some of the basic recombinant DNA techniques. These include: (1) to prepare the plasmid DNA, pUC19/tRNA2fMet (K12), from the E. coli cells (TG-1 strain) using the alkali lysis method and quantitate the plasmid DNA by spectrophotometry. (2) to characterize the plasmid DNA by restriction enzyme digestion and analyze the digested DNA fragments on agarose gel by gel electrophoresis. (3) to amplify the E. coli tRNA2fMet gene from the plasmid DNA pUC19/tRNA2fMet(K12) by the PCR technology and analyze the amplicone (220 bp) on agarose gel by gel electrophoresis.

The pUC19/tRNA2fMet(K12) plasmid contains the E. coli initiator tRNA gene fragment (220 bp). This gene fragment is amplified by PCR from the E. coli K12 strain and cloned into the Sma I site of the pUC19 vector. The sequence of the tRNA2fMet gene is shown in the Figure 1. Two PCR primers will be used in our experiment. Their sequences are:

L primer: 5-TAC GTC CGT CTC GGT ACA CCA A--3 (22mer)

R primer: 5-CAA ATC CCA CTA CGA AGG CCG A--3 (22mer)

Fig. Sequences of E.coli tRNAfMet2 gene and its flanking sequences cloned into M13mp18 vector. Box area shows the sequences of fMet2 PCR product (220 bp). The gene fragment was PCR amplified by L/R primers and cloned into the SmaI site of pUC19 vector. The flanking sequences are replaced by the pUC19 vector sequences in pUC19/fMet2 clone. The locations of other primers are also indicated.

III. References

1. Sambrook, J., Fritsch, E. F. and Maniatis, T. (eds) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press.

2. Mandal, N., RajBhandary, U. L. (1992) Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12. J. Bact. 174, 7827-7830.

3. Nagase, T., Ishii, S. and Imamoto, F. (1988) Differential transcriptional control of the two tRNA₂^fMet of Escherichia coli K-12. Gene 67, 49-57.

Fig. The scheme of pUC19/fMet2 clone. Strain for plasmid preparation is TG-1/pUC19/tRNA^fMet₂. The genotype of TG-1 is: K12, △ (lac-Pro) supE thi hsdD5/F’traD36 proA⁺B⁺ LacI^q lacZ△ M15