Introduction

DNA technology is the sequencing, analysis, and cutting-and-pasting of DNA. Common forms of DNA technology include DNA sequencing, polymerase chain reaction, DNA cloning, and gel electrophoresis. Biotechnology inventions can raise new practical concerns and ethical questions that must be addressed with informed input from all of society.

DNA Analysis Methods

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a is a laboratory technique widely used in molecular biology to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules. The following steps are used:

The temperature changes which form the steps in the PCR process are necessary as a result of the the way in which DNA polymerase works.

1. Denaturation at 92^oC-96^oC – DNA polymerase only works on single stranded template DNA, so the relatively high temperature is necessary to break the hydrogen bonds and create single stranded template DNA. (this is also why we use Taq polymerase a high temperature version of DNA polymerase).

2. Annealing at 55^oC-65^oC – the primers will not bind to the template strand at high temperatures (hydrogen bonds break at these temperatures), so cooling is required to allow the primers to bind (anneal).

3. Extension at 72^oC – this allows the Taq polymerase to work efficiently to produce replicate the DNA strand of the target DNA.

Carrying out a cycle:
a) Initially, the mixture is heated to between 92^o–97^oC to denature, or separate, the double-stranded DNA template into single strands.
b) The mixture is then cooled so that the primers anneal, or bind, to the DNA template.
c) At this point, the DNA polymerase begins to synthesise new strands of DNA starting from the primers.

Following synthesis and at the end of the first cycle, each double-stranded DNA molecule consists of one new and one old DNA strand. PCR then continues with additional cycles that repeat the aforementioned steps. Every cycle doubles the number of copies of target DNA present, because the newly synthesised DNA segments serve as templates in later cycles, giving exponential amplification up to millions of times.

Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. All DNA molecules have the same amount of charge per mass, so gel electrophoresis of DNA fragments separates them based on size only. A 'DNA ladder' is the standard made up of DNA fragments of known sizes are used to compare the results and determine their size.

Gels for DNA separation are often made out of a polysaccharide called agarose, which is heated with water and allowed to cool to create the gel. The gel wells into which the DNA samples will be placed will be created in the gel from the mould used.

The gel must be placed in a gel box, with one end of the box without the wells connected to a positive electrode, while the end with the wells to a negative electrode. The main body of the box is filled to just above the level of the gel with a salt-containing buffer solution that can conduct current.

The DNA samples are placed into the wells. One well is reserved for the DNA ladder.

The power is turned on and current flows through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they move towards the positive pole.

As the gel runs, shorter pieces of DNA will travel faster than longer ones. After the gel has run for awhile, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells.

DNA Cloning

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. The recombinant plasmid is then introduced into bacteria. Bacteria carrying the plasmid are selected and grown, and as they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.

Recombinant DNA Technology

This technology is the combining of different organism’s DNA, which could enable scientists to manipulate and alter genes to improve industrial processes and medical treatment. Transferring DNA fragments between/within species is possible because the genetic code is universal, and transcription and translation work the same in all organisms too. The first step in this technology is to produce or isolate the fragment of DNA to be recombined with another piece of DNA. There are three methods to create fragments of DNA:

1. Reverse Transcriptase

Reverse transcription is performed using PCR machine and uses mRNA rather than DNA as the starting template.

First, the enzyme reverse transcriptase uses the mRNA template to produce a complementary single-stranded DNA strand called cDNA in a process known as reverse transcription.

Next, DNA polymerase is used to convert the single-stranded cDNA into double-stranded DNA. These DNA molecules can now be used as templates for a PCR .

The value of this process is that it can be used to determine if an mRNA species is present in a sample or to clone a DNA sequence for a subsequent experiment.

2. Restriction Endonucleases

Restriction endonuclease are enzymes that cut up DNA, which naturally occur in bacteria as a defence mechanism.

There are many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences, described as recognition sequences, and therefore each enzyme cuts the DNA at a specific location.

Some enzymes cut at the same location in the double strand and create a blunt end, other enzymes cut to created staggered ends and exposed DNA bases.

The exposed staggered ends are palindromic and referred to as ‘sticky ends’ because they have the ability to join to DNA with complementary base pairs.

3. Gene Machine

DNA fragments can be created in a lab using a computerised machine. Scientists first examine the protein of interest to identify the amino acid sequence, and from that work out what the mRNA and DNA sequence would be. The DNA sequence is then entered into the computer, which checks for biosafety and biosecurity that the DNA being created is safe and ethical to produce. The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides. The oligonucleotides can then be joined to create the DNA for the entire gene. PCR can be used to amplify the quantity and to make the double strand. This process is very quick, accurate and makes intron free DNA so can be transcribed in prokaryotic cells.

Once the DNA fragment has been isolated, it needs to be cloned to create large quantities of it. This can be done in vivo using plasmids in host bacterial cell or in vitro using PCR.

Uses of DNA Cloning

Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quicklysquared.
Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function

Gene Technology in Action

1. Genetic Fingerprinting

Genetic fingerprinting is a laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the evidence came from that suspect. Conversely, if the two DNA profiles do not match, then the evidence cannot have come from the suspect. DNA fingerprinting is also used to establish paternity.

Genetic fingerprinting uses the non-coding regions of DNA that contain short, repeating sequences called variable number tandem repeats (VNTRs).VNTRs are found at many locations in the genome. In every individual, they vary in length and the in the number of repeats at different loci. Therefore, the probability of two individuals having the same VNTRs is very low. The steps in DNA fingerprinting include many of the methods we have already discussed:

Extraction of DNA & amplification using PCR (see section on DNA Analysis Methods for details)
DNA digestion using specific restriction endonucleases, leaving the VNTRs intact
Separation of DNA fragments by gel electrophoresis (see section on DNA Analysis Methods for details)
The separated DNA fragments are blotted onto a nylon sheet - this is a process called Southern blotting
The nitrocellulose paper and the probe (colourless, radioactive liquid) are placed together in a glass tube in a hybridisation oven at 65^OC to allow the probe to stick to the DNA fragments with the matching sequence. The nitrocellulose paper is then rinsed to remove any radioactive probe liquid that has not stuck. The paper will be mildly radioactive because of the probe stuck to it, so all of this stage is done in a working area set aside for radioactivity.
Development of banding pattern that can be visualised as radiation, emitted by fragments, exposes X-ray film (placed over the gel) and reveals their final positions. The radioactive probes having bound to the complementary sequences that have been run on the gel and blotted, and X-rays are used to expose the film, allowing scientists to visualise the pattern of fragments (and hence the DNA unique pattern of that individual)

2. Human Genome Project

The Human Genome Project was an international research program involving over 1000 scientists, aiming to map and understand all the genes in the human genome. This can be carried out by determining the sequence of all 3.2 billion nucleotides in the genome and characterising the features of the DNA, by figuring out which sequences code for protein-coding genes. The Human Genome Project determined there are just over 20,000 human protein-coding genes.

DNA sequencing is the process of determining the sequence of nucleotides in a piece of DNA. DNA sequencing involves cutting the DNA into small fragments, and aligning the overlapping regions, allowing the determination of the sequence of whole chromosomes and even whole genomes. During the Human Genome Project (1980s) this was complex and time-consuming job as molecular techniques were not as well developed as they are today. The scientists used a techniques developed in the 1970s called Sanger Sequencing named after Frederick Sanger who developed the technique to sequence DNA.

1. In Sanger sequencing, the DNA to be sequenced serves as a template for DNA synthesis. A DNA primer is designed to be a starting point for DNA synthesis on the strand of DNA to be sequenced. Four individual DNA synthesis reactions are performed. The four reactions include normal A, G, C, and T deoxynucleotide triphosphates (dNTPs), and each contains a low level of one of four dideoxynucleotide triphosphates (ddNTPs): ddATP, ddGTP, ddCTP, or ddTTP. The four reactions can be named A, G, C and T, according to which of the four ddNTPs was included. When a ddNTP is incorporated into a chain of nucleotides, synthesis terminates. This is because the ddNTP molecule lacks a 3' hydroxyl group, which is required to form a link with the next nucleotide in the chain. Since the ddNTPs are randomly incorporated, synthesis terminates at many different positions for each reaction.

2. Following synthesis, the products of the A, G, C, and T reactions are individually loaded into four lanes of a single gel and separated using gel electrophoresis, a method that separates DNA fragments by their sizes. The bands of the gel are detected, and then the sequence is read from the bottom of the gel to the top, including bands in all four lanes. For instance, if the lowest band across all four lanes appears in the A reaction lane, then the first nucleotide in the sequence is A. Then if the next band from bottom to top appears in the T lane, the second nucleotide in the sequence is T, and so on. Due to the use of dideoxynucleotides in the reactions, Sanger sequencing is also called "dideoxy" sequencing.

3. From the colours of dyes emitted by dye on the chain terminating nucleotide, the sequence of the original piece of DNA can be built up one nucleotide at a time.

3. GM Crops

GM is a technology that involves inserting DNA into the genome of an organism. To produce a GM plant, new DNA is transferred into plant cells. Usually, the cells are then grown in tissue culture where they develop into plants. The seeds produced by these plants will inherit the new DNA. Genetic modification of plants involves adding a specific stretch of DNA into the plant’s genome, giving it new or different characteristics. This could include changing the way the plant grows, or making it resistant to a particular disease. The new DNA becomes part of the GM plant’s genome which the seeds produced by these plants will contain.

The first stage in making a GM plant requires transfer of DNA into a plant cell. One of the methods used to transfer DNA is to coat the surface of small metal particles with the relevant DNA fragment, and bombard the particles into the plant cells. Another method is to use a bacterium or virus. There are many viruses and bacteria that transfer their DNA into a host cell as a normal part of their life cycle. For GM plants, the bacterium most frequently used is called Agrobacterium tumefaciens. The gene of interest is transferred into the bacterium and the bacterial cells then transfer the new DNA to the genome of the plant cells. The plant cells that have successfully taken up the DNA are then grown to create a new plant. This is possible because individual plant cells have an impressive capacity to generate entire plants. On rare occasions, the process of DNA transfer can happen without deliberate human intervention. For example the sweet potato contains DNA sequences that were transferred thousands of years ago, from Agrobacterium bacteria into the sweet potato genome.

There are other ways to change the genomes of crops, some of which are long established, such as mutational breeding, and others of which are new, such as genome editing

4. Gene Therapy

Gene therapy is the repair or replacement of faulty genes performed by introducing DNA containing the functional gene, to correct a disease-causing mutation. The success in gene therapy has been limited to somatic cells, which are cells are the cells that make up our body but are not inherited, as opposed to the gametes (eggs and sperm). This means that any changes made to the genetic material in a patient are not passed on to the children. Gene therapy is likely to be a promising treatment option for certain genetic diseases in which there is one faulty gene that affects a particular cell type that can be repaired.

Viruses are used as vectors to deliver the healthy gene into the cell’s genome. The DNA is expressed by the cell’s normal transcription and translation machinery, so the cell can produce the correct protein required to prevent the disease. Genes can be repaired in three ways:

1. Gene augmentation therapy where a mutation in the DNA causes a loss of the function of a gene, stopping it producing a working protein. Gene augmentation therapy adds DNA containing a functional version of this gene back into the cell.

2. Gene inhibition therapy is used in the treatment of diseases caused either by the gene being over-active or by production of a new protein that interferes with the function of another gene. Gene inhibition therapy aims to reduce or stop the activity of the problematic gene.

3. Targeted killing of specific cells is also used in cancer gene therapies. The aim is to deliver DNA into a diseased cell as before, but this time the DNA is designed as such that it will kill the cell. This can be done in various ways, and one of the most effective ways is inserting a gene that programmes the cells to die, a ‘suicide’ gene.

Ethical Issues

As technology develops ethical implications associated with it become more complex, and we need to be aware of what these may entail across the topics of human health, crime, food science and ownership of genetic information.

Food - here is a great article published in Scientific American that summarised the arguments for and against GM foods

Crime - here is a link to an article, published in the Nature journal as part of their education section, about the ethical implications of the use of DNA in forensic technology

Health - here is a link to some more great articles published in the Nature journal education section - there are some interesting topics to explore including the ethics of human genetic screening and ethics of genetic testing (genetic discrimination).