Manipulation Of DNA

Introduction

Protein–DNA interactions play a critical role in the molecular biology of all organisms. For example, the ∼3.3 billion base pairs human genome is estimated to code for at least several thousand DNA-binding proteins, including transcription factors, nucleases, repair proteins, topoisomerases, structural proteins, and DNA and RNA polymerases.

The experimental research aims to isolate DNA plasmid from an E. coli culture, undergo PCR for amplifying specific DNA fragments, perform restriction digestion using the enzymes of HindIII and XbaI followed by running the DNA fragments obtained through Agarose-Gel-Electrophoresis.

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Genetic analysis and molecular biology depend on DNA plasmid purification, following simple isolation techniques or complex ones which requires a lengthy period and high-quality preparation of genomic DNA. Manipulations of DNA by analysing through electrophoresis and through restriction digestion, needs the use of simple technique of isolation. Isolated DNA is later used in molecular biological and scientific processes like cloning, blotting, transfection, translation, PCR, restriction-digestion or transformation. Plasmid Isolation from cell-suspension solution of bacterial cell often and mostly widely uses the process of alkaline lysis, where the bacterial cell is broken down by a detergent. Other reagents used are alkaline-lysis-buffer and strong NaOH. The detergent is used for cleaving phospho-lipid membrane bi-layer and alkali denatures the protein, breaking the structure of the membrane. This is followed by sequences of agitation process, precipitation, centrifugation and supernatant removal. Finally, by removing cell-debris plasmid is purified and thus isolated.

Systematic Procedure

Potassium acetate is also added for pH neutralisation. During centrifugation, SDS is made insoluble, by interaction with Potassium ions which precipitates and is separated. DNA is in the procedure, trapped by SDS and removed from supernatant leaving plasmid DNA and RNA in solution. RNaseA digests the RNA removing it, leaving RNA nucleotides, protein and carbohydrates only in the solution. Ethanol precipitates DNA by adding water causing clumping of DNA. Thus, insoluble purified DNA is extracted in pellet form.

The Polymerase-chain-reaction or PCR process amplifies and makes multiple copies of target DNA fragments in vitro, using Taq polymerase (thermostable), oligo-nucleotide-primers (2), Mg2+, reaction-buffer and dNTPs with cyclic repetitions of temperature within a few hours of time (Palacio-Bielsa, et al., 2011). The thermal cycler, used in PCR amplifies target DNA through temperature variable and time variable steps of template-denaturation, primer-annealing and extension, causing amplicon doubling. Denaturation takes place at about 94°C (2 minutes) followed by annealing for 60 seconds at 40-60°C and extension (2 minutes) at 70-74°C for completing a single cycle.

Following the Agarose-Gel-Electrophoresis, the polysaccharide Agarose is used, and the fragmented DNA are visualised for experimental use. A DNA molecule with similar charge-mass-ratio, mobility does not get influenced on account of electrostatic charge (Lee, et al., 2012). DNA fragments of different size and different conformation moves across the gel at different rates. Once the electricity is switched on, the motility of DNA species through Agarose-gel follows inverse proportionality with molecular sizes (Huang, et al., 2010). The larger fragments fail to travel longer distances remaining closer to the well. Also, conformational differences are used for DNA differentiation. Super-coiled and nicked DNA moves slower than linear DNA.

Plasmid-DNA purification

The isolation/purification of DNA has been conducted according to the protocol of Gomes, et al., (2009). E. coli cells (5-10 ml TOP10 pUB01) were transferred to culture-tubes over-night. This was then centrifuged and supernatant discarded. 250μl resuspension-buffer or P1 was used for resuspension of pellets following transfer to 1.5ml microfuge-tube. A lysis-buffer or P2 of 500μl was then added and intermixed via inversion. After this neutralisation-buffer or P3 (600μl) was added. This too was inverted repeatedly for mixing. Centrifugation pelleted the debris and sample was then transferred to collection tube followed by another centrifugation for 1min. Filtrate was then discarded, reusing collection tube. In Spin column the residual sample was taken repeating the last step one more time. 500μl wash-buffer (pre-heated to 50°C was added following centrifugation. After discarding filtrate so obtained, wash-buffer (600μl) or PW2 was added and re-centrifuged for 1min after which filtrate was again discarded and centrifuged for 2min at maximum speed. Collection tube was discarded and was placed in 1.5ml Elution tube. Now Buffer P (60μl) was heated to 70°C in the spin-column-membrane. This was then incubated for 2 minutes and DNA was eluted for 1 min at 11,000 x g speed to obtain isolated DNA storing it in sub-zero temperature.

Agarose-Gel-Electrophoresis

Agarose Gel preparation – The protocol followed for gel preparation was in respect to Lee, et al., 2012). 1% of agarose-solution was initially prepared in TAE electrophoresis buffer with 1X strength, boiling it and the cooling for uniformly dissolving the powder. 1g/ml of Ethidium bromide was then added, and comb placed at the system. The Gel was poured at a thickness of 5mm, cooling it for 30min for gel to set.

6 reactions were analysed consisting of 2 PCR and 4 RE reactions in the electrophoretic system. The protocol enlisted in Lee, et al., (2012) was followed and 1x conc. gel-loading buffer along with bromophenol blue dye was then added to the samples. 2 µl gel-loading buffer upon added to 2 PCR reactions each, was made to 10 µl. 4 µl of gel-loading-buffer upon added to RE were made to a total 20 µl each. 7 µl of molecular-weight-marker was then loaded upon gel, comb was then removed TAE (1 x conc.) was added to gel tank until submerged. Pippeting of sample was done and sample loaded in order of:

Results

The Agarose gel annotated image is estimated according to the DNA fragment sizes obtained from the RE digest and PCR analysis. The plasmid map (give below) is then used for understanding the size of DNA insert located between the two TOPO Recognition sites. Furthermore, the distance between forward and reverse primer, between XbaI and Hind III recognition sites were also analysed.

According to the Agarose Gel-electrophoresis results, it can be seen that the 1st lane represents the standard DNA-molecular-weight marker, lane-2, shows no-DNA Polymerase-Chain-Reaction, lane 3 represents the reaction of pUB01 PCR, lane 4 represents pUB01 no enzyme digest control; lane 5 represents pUB01 XbaI digested product; lane 6 represents pUB01 HindIII digested product and finally the lane 7 represents pUB01 XbaI and pUB01 HindIII digested product.

The molecular-weight of DNA ladder was 10 kilo-bases and was considered as standardised DNA molecular-weight-marker. While the pUB01 PCR reaction shows no band, the HindIII Digested reaction, XbaI digested reaction and the pUB01 XbaI + pUB01 HindIII digested reaction shows DNA bands. The band obtained from XbaI digested reaction, is of about 8 kb; the bands obtained from pUB01 HindIII digested reaction is also 6 kb in size, while the bands obtained from the pUB01 XbaI + pUB01 HindIII digested reaction are of 6 kb and 1 kb, according to the DNA size standard.

Discussion

In the result, DNA bands were obtained in the restriction endonucleases digestion reactions of XbaI digestion and HindIII digestion as well as in XbaI + HindIII digestion. 4 series of RE digest was set with the substrate pUB01 DNA and Restriction-Enzymes of XbaI and HindIII and set for 1hr incubation at a temperature of 370C. The protocol that was used mostly followed the widely used RE-digestion protocol of Bryda, and Bauer, (2010).

The protocol followed in the PCR is that of Garibyan, and Avashia, (2013). However, no bands were obtained in the agarose-gel in the lane-3 PCR reaction. This is similar to the research of Lorenz, (2012), wherein PCR trouble-shooting or a lacking DNA band after PCR reaction resulting is likely to occur because of stringent reaction conditions namely hairpin-loop formation, primer dimmers along with primers or even in denatured DNA template. This therefore prevents PCR product amplification as such molecules fail to base-pair with DNA counterparts. According to Boesenberg-Smith, et al., (2012), DNA assessment and yield through PCR often overlooks certain important aspects in laboratory preparation and implementation of the PCR process, leading to PCR troubleshooting. Some other factors may be thermal-blockage of the PCR-system, wherein the heat-generator inner-tube-holder was not functioning. A high annealing-temperature may also be another issue, wherein the primers will not be bonded to template DNA. If the primers have faced degradation, or the template DNA due to inappropriate storage temperature like longer room storage can change primer or template DNA integrity. If PCR inhibitors are present in template DNA, then that too may be a possible explanation to not obtaining a PCR reaction band. EDTA and ethanol may cause this. The failure in obtaining a band in PCR may be because of several reasons like forgetting to add any component during the practical experiment, using wrong PCR condition by incorrect programming, and wrong-cycle conditions applied in the cycle. Another possible causal factor could be the fact that Taq DNA-polymerase was not functioning. Contamination with nucleases from skin or dirty bench, could have entered reagent and destroyed template. Primer-design may also not have been suitable to the template. All these may be reasons behind the failure of obtaining PCR bands in the reaction.

References

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