There are many steps in the process of genetic manipulation of DNA from cutting it to amplifying the recombinant molecule which has been produced.
In order to isolate biochemically useful DNA the sequence should be as long as possible. The DNA must first be obtained from the required cells. If eukaryotic cells are being used this is a relatively simple process as they cells are surrounded by only a lipid membrane. Lipid is soluble in detergent and so adding detergent to eukaryotic cells will lyse them. A commonly used detergent is called sodium dodecyl sulphate (SDS).
Enzymes such as nucleases, which lead to the degradation of genetic material also first, have to be inactivated and SDS does this too.
When using prokaryotic cells the polysaccharide coating has to be first removed using the enzyme lysozyme, then the SDS can be used to lyse the cell etc. Insoluble material is removed by centrifuge leaving the required genetic material, DNA (and RNA) along with protiens.
When DNA has be removed, the genetic material has to be purified, for genetic material of high molecular weight, Phenol can be added to the insoluble solution that has been obtained. This is mixed which creates an emulsion of all the tube contents.
Centrifugation of this will lead to the formation of two layers, with the genetic material in the upper phase, the phenol in the lower and the insoluble protein on the interface between the two layers. By removing the uppermost layer the genetic material can be successfully removed. This upper layer which has been removed still contains RNA, which can be separated from the DNA by "buoyant density centrifugation". In this process the genetic mixture is placed in to a centrifuge tube with Caesium chloride at high concentration. Once a gradient is set up the concentration in the centre of the tube will enable the DNA to concentrate there by equilibrium. This tube is centrifuged for up to forty hours at 40,000 rpm. At such a high speed the centrifugal forces imparted in to the solution, will cause the heavy caesium ions to move towards the outside of the tube. This causes density and concentration gradients to be set up, giving rise to the DNA (which has a slightly lower density than RNA), to concentrate towards the middle (due to the differing sedimentation coefficients of the molecules). The denser RNA moves towards the sides of the tube (or the bottom depending on tube orientation).
The DNA obtained can be processed in slightly varying ways. If a high molecular weight is not required then adding a large concentration of ethanol, which is then centrifuged to form a pellet, can precipitate the DNA. The remaining ethanol is then removed by drying and the remaining pellet is re-dissolved in the required buffer. For situations where large DNA fragments are required, the DNA is not precipitated using alcohol, as the high pressures imparted by the centrifugation can lead to the molecules being sheared.
'Restriction endonucleases' or 'Restriction enzymes' are the backbone behind recombinant DNA technology. The power of these enzymes lies in their ability to cut DNA in to defined fragments at specific sequences of the genetic coding.
Restriction enzymes originate from many different forms of bacteria. It had been noted that some forms of bacteriophage grew in forms of E.coli but not in apparently similar forms. It was found that these bacteria were producing an enzyme, which had the ability to digest DNA at specific sites on DNA, another complimentary enzyme was found that had the ability of methylating the bacterial DNA at the same site. This methylation protected the bacterial genome from being digested by an endonuclease it produced, but the endonuclease still degraded any unmethylated DNA in the bacterial cell such as that of invading viral DNA, this is known as 'host restriction'. Many restriction enzymes have been found, in many different bacteria, all of which have the ability of protecting bacteria against viral DNA being integrated in to its own genetic sequence. Each of these enzymes recognised and 'restricted' a different and specific genetic sequence.
This wide range of different enzymes (and therefore different cutting sites) enables us to manipulate DNA in such a simple yet specific way. Apart from each enzyme cutting at a different specific site, another property of these endonucleases is their differing properties in producing cutting sites. Whilst some leave 'blunt' ends with no DNA overhang others produce cohesive or 'sticky' ends which aid in the re-ligation of genetic material. Although some enzymes such as BamH and BglII recognise different cutting sites, the cohesive ends they produce are both identical, which may be a useful property if engineering with more than one enzyme or genetic insert. The names of these endonucleases are determined from the bacteria from which they are obtained; for example EcoR1 is derived from E.coli. The figure 1 in EcoR1 indicates that this was the first enzyme to be isolated from E.coli.
The most important qualities of restriction enzymes are their digestive properties. Most commonly used enzymes recognise a short sequence of bases (usually 4-6 bases long). The number of bases in a recognised sequence is important as if an enzyme recognised just one base i.e. adenine, then on average the DNA would be able to be cut at every fourth base. For any sequence of six bases to randomly occur the enzyme would statistically only find a cutting site every four thousand and ninety six bases. Although the sequence of bases in genetic material are not randomly distributed as genes are related and sequences conserved this specific site recognition lowers the number of cutting sites on a genome considerably. On a bacterium with a small chromosome, there may only be one or two such cutting sites. When an enzyme such as EcoR1 cuts the DNA, a phosphate group stays on the 5' end of the DNA and an OH group resides on the 3' end. If the cut in the DNA does not create blunt ends, the ends of the DNA are regarded as sticky because of their ability to briefly pair again, however at normal room temperature this pairing is unstable and short lived.
Once DNA has been restricted, using an enzyme or number of enzymes, each fragment that has been created may be of an unknown length and the total number of fragments may be unknown. To resolve these problems a technique named 'agarose gel electrophoresis' is employed. The agarose gel used in this technique has the property of becoming a liquid when heated and setting in to a jelly like substance when it cools because of the cross-linking of large polysaccharide molecules in it. By setting up a difference in voltage across the gel, it is possible to impel fragments of negatively charged DNA molecules to move through the agarose, away from the cathode and towards the positively charged anode. Because of the cross-linking in the gel, the larger molecules have a greater resistance towards moving through the gel than the small ones so they progress more slowly. The distance the DNA has moved is measured from the well it was loaded in to (at the cathode end), to the centre of the band at the end of the run.
Because different DNA and different enzymes can yield fragments of drastically varying size, it is important to separate out the bands as much as possible without losing any or letting any merge.
To do this the run can take place using currents of different strengths and by using agarose which has different amounts of cross-linking so giving different specific resistance's to the material travelling through it. This process helps to clarify each band and to obtain accurate, measurable results. When the DNA is pipetted in to the wells, it goes in with a running dye, which will also run towards the anode. Due to its size it will move faster than the DNA so when it reaches the end the run can
be terminated and no bands should have been lost.
Once the run is complete, it is important to visualise the positions of the bands of the genetic material. The gel is removed and placed in a bath of ethidium bromide; this substance has both the property of fluorescing under ultra violet light and of getting between the bases on a DNA molecule. The DNA in the gel will incorporate the ethidium bromide and so fluorescent bands on the gel will determine their positions when exposed to UV light. These band positions are recorded and used to determine the size of each fragment. As ethidium bromide gets between the bases of DNA it is a potential mutagen and therefore a potential carcinogen and exposure to it should be kept as low as possible. The mobilities of DNA fragments of a determined size are plotted against the log10 of their size. By this method the size of unknown fragments can be determined by reading the distance they have moved against the equivalent log size on the graph.
As well as cutting DNA using endonucleases, an important aspect of genetic manipulation is the ability to join fragments of genetic material together. The cohesive ends that remain after the digestion with restriction enzymes are naturally complimentary, but are inherently unstable under normal conditions and will not rejoin easily. By using the enzyme 'DNA ligase', the cohesive ends left by the digestion with the enzyme will be able to join again creating one large fragment rather than many smaller ones. The ligase enables the 3' OH group to covalently bond to the 5' phosphate group re-ligating the molecule together. This will only work if the vector DNA and the DNA have both been digested using the same enzyme, or enzymes that produce the same sticky ends. Ligase is often obtained from E.coli, which has been infected with the T4 bacteriophage, this requires ATP as a co-factor but the T4 ligase produced has the ability to join blunt ends well.
Now that the recombinant DNA ligated in to a suitable vector such as a plasmid or bacteriophage it is important to introduce it in to a system that will amplify it, such as a bacterium.
Under normal conditions bacteria will not take up these free plasmids and have to become 'competent' to do so, this process is known as 'transformation'. Treating the bacteria with a solution of calcium chloride and then 'heat shocking' does this. The plasmid and bacterial solutions are mixed and some of the bacteria will take up the plasmids. As less than one percent usually do so; each bacterium should take up a maximum of one plasmid and should only contain one recombinant molecule. By using a plasmid containing a gene that codes for resistance to an antibiotic such as the Amp gene (which gives resistance to ampicillin), it can be determined which bacteria have taken up a plasmid. The bacteria are plated out at low concentration on an agar jelly containing the said antibiotic, and if none of them had resistance before the treatment only the ones containing the plasmid and resistance gene will survive. As each bacterium forms a colony, each cell in it will contain a single type of DNA molecule; this process is known as DNA cloning as each cell in a colony is identical to the rest.
Increasingly there are many uses for such genetic manipulation, by inserting the gene that codes for human insulin in to bacteria, they will begin to create the insulin protein and provide a useful drug for those with diabetes. Another approach to genetic engineering is the manipulation of human somal cells, such as in 'gene therapy'. Sufferers of cystic fibrosis have inherited two copies of a defective gene that should control the mucous glands mucous production leading to a build up phlegm on the lungs. An experimental approach to dealing with such a situation uses a cold virus, which has been genetically manipulated to deliver a working version of the gene to the sufferer's lungs. This gene should then begin to code for the protein that is normally not produced by patients with cystic fibrosis.
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