Cloning DNA with PCR
If we have obtained a DNA sample or a few, what next? Well, nothing much can be done with that. We must obtain exponentially more DNA to use for any purpose. And it all of course must be identical. We must essentially clone our DNA. Considered the very staple of molecular biology, this technique for multiplying DNA many-fold was invented by a chap Kary Mullis who believes in astrology.
Essentially the DNA is denatured so the 2 strands break apart, short complementary bits called primers attach to the strands, the enzyme DNA polymerase binds to the primers and initiates the assembly of a new DNA strand, and finally the process is repeated many times over in a chain reaction. This is the polymerase chain reaction, PCR.
Soon enough, the few bits of DNA become thousands, and hundreds of thousands, and millions…
Visualising DNA with gel electrophoresis
A common method of visualising differences is gel electrophoresis which involves loading small volumes of samples on a gel and running a current across it in order to separate the samples by size.
Since the gel has a microscopic matrix inside that provides resistance against sample movement through it, the larger molecules move more slowly while the smaller fragments can move more quickly.
The positive charge is at the bottom of the tank, while the samples are loaded at the top. This way, they will move downwards towards the bottom of the gel because they have a negative charge as molecules. The current is run across the gel for around 30-60 minutes (ensuring the samples don’t run too long and hence run off the gel into the buffer solution! if that happens they are lost) after which the sample’s progression on the gel can be visualised by using a stain solution or pre-existing coloured label visible under UV light.
Genetic fingerprinting technique
1. The sample DNA undergoes PCR then cleavage at multiple sites with restriction endonucleases (enzymes that cut DNA at specific sequence locations)
2. The resulting many small fragments are tagged using a radioactive molecule
3. They’re separated using gel electrophoresis and viewed using a developed photographic film
The bands exposed then undergo simple visual analysis by matching up the template DNA with other DNA that could be similar more or less, depending on situation. Above, the DNA found at a crime scene is compared with that of 3 suspects. The bands of suspect 2 are perfectly aligned with the crime scene DNA.
Say we are interested in the gene for insulin. Sure, we could take it straight from people, but remember humans are eukaryotes and eukaryotes have non-coding sequences within their genes called introns. The mRNA following splicing, on the other hand, has no introns! How can we make DNA from mRNA?
Take this arbitrary bit of mRNA: UCCAUGCCAUUUGGG
If we had an enzyme which could reverse the transcription back into DNA, this time intron-free, that would be great. We do – it’s called reverse transcriptase and it produces DNA. This special case of DNA is called complementary DNA – cDNA.
cDNA via reverse transcriptase: AGGTACGGTAAACCC (remember that DNA unlike mRNA is double-stranded; not shown for simplicity)
If we wanted the portion after the second G above, is there a way we could cut the DNA? It appears so. Some microorganisms have actually evolved enzymes whose job it is to invade a host and chop its DNA up at specific sequences. These enzymes are called restriction endonucleases. Each has its own short sequence which it recognises. There is a restriction endonuclease called CviQI which has the recognition site GTAC and cuts between G and T. That fits our bill!
The other DNA strand will also have a GTAC site read in the opposite direction. Notice that the complementary sequence of GTAC backwards (starting from the C) is… GTAC! This is called a palindrome and all restriction endonucleases will have one simply by the virtue of DNA bases being complementary.
Another method of synthesising DNA called oligonucleotide synthesis involves the chemical manipulation of the nucleotides by adding each at a time to the growing chain. Due to the high error rate of this technique, the length of the oligonucleotide is limited to around 200 bases. Most often, much shorter sequences are used around 15-25 base pairs for the purpose of cloning DNA (see below), interference RNA, use as probes in various tests, etc.
The outline of oligonucleotide synthesis shows how the nucleotides are chemically modified throughout the chain-lengthening process. The overall reaction switches from solid-state to liquid state at the end.
Cloning DNA like this in vitro (outside a living organism) is a quick and reliable method of obtaining large amounts of DNA of interest. This is useful in criminology for DNA identification, for the study of various organisms by analysing their DNA, etc.
Designing the new DNA fragment to be cloned involves the addition of some basic, often standardised elements that enable the expression the gene. Two important ones are the promoter and the terminator. As the names indicate, these parts are found before and at the end of the gene and control the start of transcription of that gene, as well as the end of it.
In vivo (inside a living organism) gene cloning involves stimulating bacteria to take up target DNA by inserting it in a circular bit of DNA they normally carry beside their “main” DNA, called a plasmid. This is often transferred horizontally between bacteria, and always passed down through the generations. Target DNA may be inserted in plasmids via PCR when restriction endonuclease leave sticky ends which can be joined back.
Plasmids also contain an antibiotic resistance gene which, if taken up successfully by bacteria, will enable their growth on a medium containing that antibiotic. This allows the selection of only bacteria which have taken up the plasmid (vector), and with it our DNA of interest.
The host cells (bacteria) can now be grown on a large scale. They will express the new DNA in the plasmid, and pass it on to their offspring cells to do the same. Shortly, there will be a massive number of bacteria producing whatever the gene of interest codes for. This could be human insulin.
The product can then be isolated and used. It is in a pure form and is in fact being used worldwide to treat Type I diabetes. This breakthrough enabled better management of the condition which had previously been treated with non-human insulin which had side effects.
Genetically modified organisms (GMOs)
Recombinant DNA technology can bring with it new ethical, financial and social issues. For example, some people decide to patent basic DNA such as that of certain crops, and charge farmers to grow those crops.
In medicine, gene based treatments can also be severely restricted financially. The interest of people who want to extort new technology for their immediate profit results in a huge burden of treatment cost to patients or national health services.
Social issues include genetic modification of plants, animals and human embryos. The ability to pick or remove various traits associated with key concerns in society such as appearance, function and diversity can pose conundrums. Three-parent babies? GM food? Heritable deafness as a disease or valid option belonging to a strong community and culture?
Super soya beans
The first genetically modified (GM) soya beans were introduced by Monsanto in 1994. They now occupy the majority of soya land.
The applications of this technology focused on increasing the yield of soya beans at as low a cost as possible. In time, it became apparent that various other elements could also be improved, as the use of soya spans many different products. The soya beans could be made healthier and more valuable by adding several genes foreign to itself, from bacteria and other plants (Roundup Ready Soybean). These can be delivered using a gene gun.
DuPont Pioneer is a company that developed a GM soya bean that makes the resulting soya oil more valuable. Naturally, the soya oil is very susceptible to oxidation and hence making the oil rancid. By silencing or knocking out the delta 9 and delta 12 desaturase enzymes, they made a soya bean with an altered fatty acid composition high in oleic acid and stearic acid, and low in linolenic acid. This different fatty acid profile makes it less susceptible to oxidation.
Although the scientific community has concluded that GM food is equally safe to eat as non-GM food (as tested individually for each new GM food to be marketed), public opinion is still relatively against it. GM food sparked a big debate around its advantages and disadvantages to big agriculture companies, consumers, scientists, farmers and others.
On one hand, GM food improves yield and the overall value extracted from crops. This is significant to the economy as well as areas where people struggle to have enough food to eat. Companies that develop GM food sell seeds and get to make large profits which can be used to further research GM food.
On the other hand, patenting food raises issues for small farmers in developing countries, as well as other ethical concerns of big companies owning the food source of the world. Suspicious consumers also don’t fully trust GM food, so may choose to avoid it in favour of non-GM food.
These concerns that affect multiple parties constantly vie for attention, and form the dynamic of attempting to accomplish balance in this debate.