Report Discussion

Discussion:

Our objective was to characterize and sequence an unknown, experimentally obtained segment of genomic DNA from Bactrocera dorsalis. The segment was subjected to several common techniques of genetic analysis (genomic library, amplification of segment in vectors, restriction enzyme digest, bioinformatic analysis of segment, Southern blot to verify inserted sequence, and PCR amplification).

Genomic DNA obtained from the fruit fly Bactrocera dorsalis was treated with EcoRI, which cleaved the genome at each G⇓AATTC recognition site, producing randomly sized, unique DNA fragments. Which were ligated into EMBL3 bacteriophage-λ vector. Once the B. dorsalis fragments were ligated into EMBL3, the recombinant phage was allowed to infect Escherichia coli cells. Once inside the host E. coli cells, the phages replicate by subverting the host’s replication machinery, as phages are not capable of replication outside the host. In this process the bacteriophage DNA may be incorporated into the host cell’s circular DNA strand, as occurs during the lysogenic pathway. If the proper induction event occurs, the cell can be triggered to enter the lytic pathway. During the lytic pathway virus-specific enzymes are created, which allow many replicates of the bacteriophage DNA to be packaged for the infection of other cells. The cell then lyses, and the bacteriophages are released from the cell. When they come in contact with nearby bacterial cells, they land on the cell surface and inject their DNA into the cell cytoplasm. This reinitiates the infection sequence. When many cells are lysed as a result of a series of infections by replicates of one phage particle, a plaque is formed.

The presence of plaques on the bacterial lawn indicates that λ-phage infection was successful. The 45 plaques compose the genome library, and each plaque corresponds to one clonal population of λ-phage with a unique inserted segment of genomic B. dorsalis DNA. One of these plaques was selected for further analysis. The phages present in the plaque were again treated with EcoRI to separate the B. dorsalis DNA from the EMBL3 backbone. The MOI (multiplicity of infection) used in this experiment was far below a 1:1 ratio. If a 1:1 ratio were used, there would be one phage for every one bacterium. All, or nearly all, of the bacteria would die as a result of infection, and no bacterial lawn would be present. The entire agar surface would be plaques of lysed cells, and it would be too difficult to recover one amplified fragment of inserted DNA.

Ligases are enzymes that join the ends of DNA fragments by creating sugar phosphate bonds between the two ends. A vector plasmid (pUC19) containing the lacz gene that produces the enzyme beta-galactosidase and the ampr gene produces enzymes that confer resistance to the antibiotic ampicillin to the host cells. The enzyme beta-galactosidase cleaves X-gal, a synthetic substrate added to the growth medium, and produces products that have a blue color. The restriction enzyme cleaving site (multiple cloning site) is located in the middle of the lacz gene, however. If the foreign DNA fragment is appropriately inserted in this region by way of ligation, the gene will be interrupted and not properly function. Beta-galactosidase will not be produced, and X-gal will not be cleaved to produce a blue color. The colonies will appear white. Also, the presence of colonies confirms the uptake of the plasmid, which contains the ampicillin resistance gene. Without the plasmid, the bacterial cells would not grow on the medium containing ampicillin (LB-amp-Xgal).

476 blue colonies and 512 white colonies were observed on the selective LB-amp-Xgal medium. The presence of colonies on the plate indicated the positive uptake of the plasmids by E. coli cells, although not all of the plasmids contained the recombinant DNA (some were merely pUC19). The white color was indicative of tranformants that contained plasmids with inserted DNA, as the lacz gene was interrupted. The blue color indicated that transformants contained plasmids, but the DNA insert did not interrupt the lacz gene (as these cells still produced beta-galactosidase).

The most likely explanation for the continued production of beta-galactosidase is that B. dorsalis DNA was not inserted into the plasmid, but it is also possible that the DNA was ligated into another region of the plasmid vector (unlikely). A blue colony could occur if the insert DNA adjacent to the longer sequence of the interrupted beta-galactosidase gene was sufficiently similar to the sequence that had been there before cleavage and ligation. The gene might still properly function, cleaving X-gal into blue products. It is also possible that the insert DNA could contain the beta-galactosidase gene.

Restriction enzymes, which cleave DNA at specific sequences (recognition sites), can help characterize unknown DNA segments. By treating a sample of DNA with a combination of single and double digests, a restriction enzyme map can be created, indicating the approximate position of known recognition sites along the target sequence.

Restriction enzymes were selected that did not have recognition sites in the pUC19 vector (other than MCS) in order to facilitate map creation. Bam HI, EcoR1, HindIII, and Pst1 had recognition sites in the MCS of pUC19. Xho1 did not have any recognition site in the pUC19 vector.

After electrophoresing the DNA fragment products of restriction enzyme digests, the sizes of each fragment were calculated by comparison with the 1kb marker. A restriction enzyme map of the recombinant plasmid was constructed. The pUC19 backbone is approximately 2700 bp in size. The recombinant plasmid had a size of 8700 bp as indicated by the fragment sizes of BamHI and HindIII digests, which both cleaved the circular plasmid only once. The insert DNA was around 6000 bp. The HindIII restriction site was located around 500-bp from the MCS, the Xho1 restriction site was around 1000 bp from the MCS, and the Pst1 restriction site was 3550 bp from the MCS. EcoR1 restriction sites occurred at both interfaces of the pUC19 vector and the B. dorsalis DNA insert. This constructed restriction enzyme map was compared to a computer-generated map (Figure 4) for 720-bp of the insert sequence. A HindIII restriction site was found at bp 175 of the 720-bp sequence. This indicates that the 720-bp sequence did not originate at the MCS, but approximately 545 bp distal to it. The Xho1 site was not seen in the 720-bp sequence.

Probes and Southern blots are useful tools in their ability to verify the presence of a specific DNA sequence. A probe can be constructed that matches the target sequence. A template strand that matches the target sequence is amplified using PCR. Initially, denatured sample DNA is dotted onto a nylon membrane and crosslinked using UV radiation. Next, prehybridization is performed by wetting the membrane and blocking sites that could allow non-specific binding by the probe. Following prehybridization, the denatured probe DNA is added to the membrane-bound samples. Hybridization occurs between the probe and its complimentary target sequence. Following hybridization, the samples are washed several times in order to remove non-bound and weakly bound probe sequences. Following the wash sequences, the specific binding of the probe to the target sequence can be detected.

The mechanism of detection of DNA sequences using the Southern blot method is simple, although there are several steps. Initially, a probe is created that contains the nucleotide sequence under investigation. During hybridization, this probe sequence will bind complementarily to the target sequence if it is present in the sample. A “tag” or label is necessarily built into the probe to verify hybridization with the target sequence. One type of tag involves the use of DIG-UTP probe, in which a modified nucleotide (DIG-UTP) is used to synthesize the probe during PCR. Once the probe hybridizes to the target sequence on the membrane, the sample is treated with an enzyme-bound (alkaline phosphatase) antibody that is directed against the DIG modification. The antibody and the enzyme adhere to the DIG-UTP nucleotides. When enzyme-specific substrates are added to the sample, the presence of the target sequence can be verified by formation of the products, which often designed to yield a change in color (in this case blue).

A dot blot was conducted on the experimental recombinant sample in order to verify the presence of the actin-coding gene in Bactrocera dorsalis. After ligation and cloning of the insert in the pUC19 vector, verification of the target sequence is necessary to validate the results obtained. The results indicated that a blue coloration was produced in the sample containing recombinant DNA. The presence of the target sequence was verified, by cleavage of the added substrates and the formation of blue products. This cleavage was performed by the enzyme conjugated to the antibody, which was directed toward (and adhered to) the DIG-UTP nucleotides present in the previously synthesized probe.

The negative control sample did not produce any blue coloration. This indicates that cleavage of the substrates did not occur, and therefore, the enzyme responsible for cleavage was not present. All of the enzyme-conjugated antibodies were washed off, as there were no DIG-UTP nucleotides present in the sample. The probe did not bind to any sequences in the sample, and the target sequence was not detected in the pUC19 vector. It can also be concluded that no non-specific binding occurred by the probe or the antibody.

Polymerase chain reaction (PCR) amplification is a convenient method of amplifying a section of DNA in vitro. In contrast to in vivo cloning of a plasmid containing a ligated DNA insert, PCR can produce billions of copies of the target DNA sequence in a very short time with little effort required for isolation of the sequence after amplification. The standard cycle of PCR amplification involves the repetition of three steps: denaturation (during which the DNA strand is denatured to produce two template strands), annealing (during which pre-selected primers bind to the target sequences), and extension (during which the DNA polymerase elongates the DNA strand through complimentary base pairing of the dNTPs provided in the reaction mixture). The key to the PCR process is the use of a heat-stable DNA polymerase. Taq polymerase, isolated from Thermus aquaticus, is heat-stable, and therefore survives the denaturation stage of the cycle.

The presence of clearly visible, high-intensity bands in the lanes containing the experimental DNA (lanes 3, 5, 9, and 11) indicates that some sequence of DNA was amplified by the PCR process. It also indicates that the primers, polymerase, and dNTPs functioned properly and the buffer and MgCl2 concentration were suitable for amplification. Based on the migration distance of the band, which was greater than the 200-bp marker and less than the 100-bp marker, it can be concluded that the amplified sequence was between 100 and 200 base pairs in length. This corresponds to our hypothesized amplified segment length of 161 base pairs (this length was determined by the selection and design of primers 1 and 2). The results were positive, indicating the target sequence was present and sufficiently amplified.

The electrophoresed negative control samples that contained only the PCR amplification mixture and 0.5 µl of H2O in place of DNA (lanes 4, 6, 8, and 10) produced no distinct bands. This indicates that no sequence of DNA was amplified during the PCR process. If the sample had been contaminated by foreign DNA, this DNA would have possibly been amplified (if it contained a sequence similar to that of primers 1 and/or 2 or if those primers bound non-specifically) as indicated by the presence of a band in the electrophoresis gel. The negative control produced no results, indicating no significant contamination.

In conclusion, these techniques were combined to characterize and confirm the unknown sequence of B. dorsalis DNA as a gene that codes for the protein actin, a highly conserved protein among species that functions in the cytoskeleton and in muscular contraction of B. dorsalis cells.