DNA TECHNOLOGY

Some uses include treatment of human disease that are unable to do certain metabolic reactions, mostly due to unable to produce a certain protein due to a mutation of a DNA sequence. For example, insulin in Type I diabetics. Previous methods was extracting from human or animal donor, however, this had problems of rejection by the immune system, rick of infection and cost. A more modern method is to produce large quantities of these proteins using a transgenic or genetically modified organism (GMO) with recombinant DNA – DNA that is made of combination of two organisms. This is possible as the genetic code and the mechanism of transcription and translation are universal across all organisms. This is an indirect evidence of evolution.

The process of making a protein using DNA technology of transgenic organism:1. Isolation of the DNA fragments that have the gene for the desirable protein2. Insertion of the DNA fragment into a vector3. Transformation – transfer of DNA into suitable host cells4. Identification of the host cells that have successfully take up the gene by use of

gene markers5. Growth/cloning of the population of host cells

There are three methods for isolation of DNA:

Conversion of mRNA to cDNA using an enzyme reverse transcriptase that can be found in retroviruses. The enzyme catalyses the production of DNA from RNA.

A cell that readily produced the protein is selected These cells have large quantities of relevant mRNA, which can be more easily

extracted than relevant DNA. Plus human DNA contains many non-coding DNA sections and mRNA would not have it.

Reverse transcipatase is than used to make a single-stranded DNA from RNA. This DNA is known s complementary DNA (cDNA).

To make double-stranded DNA, DNA polymerase is used.

Restriction endoculeases to cut DNA of a desired gene into fragments. These enzymes come from bacteria that have develop them as a protection from viruses. There are different types, each oen cuts the DNA double stand at a specific sequence of bases, recognition sequence. This can be between two opposite base pairs leaving tow straight edges, blunt ends. Some can also cut in a straggered fashion, uneven, with some of DNA bases at the ends being exposed, known as sticky ends. In genetics, the term palindrome refers to a sequence of nucleotides along a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) strand that contains the same series of nitrogenous bases regardless from which direction the strand is analyzed. Akin to a language palindrome—wherein a word or phrase is spelled the same left-to-right as right-to-left (e.g., the word RADAR or the phrase "able was I ere I saw elba")—with genetic palindromes it does not matter whether the nucleic acid strand is read starting from the 3' (three prime) end or the 5' (five prime) end of the strand.http://science.jrank.org/pages/4995/Palindrome.html

Gene machine:1) The desired genetic sequence is determined from desired protein. Amino acid

sequence is determined. mRNA codons and then the DNA sequence are found out.

2) The desired sequence is fed into the computer. It is checked for biosafety and biosecurity to make sure it meets international and ethical requirements.

3) The computer designs a series of oligonucleotides (small, overlapping single stands of nucleotides) from which the gene can me assembled. It is an automated process, a nucleotide is added at a time.

4) Then the oligonucleotides are joined together to make a gene and then replicated using the polymerase chain reaction. It constructs a complementary stand to make the DNA double-stranded and then multiplies the gene to produce many copies.

5) Using sticky ends the gene can be inserted into bacterial plasmids. 6) Genes are checked using standard sequencing techniques and those with errors

are rejected. The advantages of this method is that it is rapid and produces DNA without introns and other ‘non-coding’ DNA.

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The sequences of DNA that are cut by restriction endonucleases are called recognition sites. If they cut in staggered fashion and leave ‘sticky ends’. Obviously the two sides created when cut are complementary to each other. Due to specificity where the enzymes cuts, if the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complementary to one another. This means that the single-stranded end of any one fragment can be joined to the single-stranded end of any other fragment. Once the complementary bases of two sticky ends have paired up, an enzyme DNA ligase is used to bind the phosphate-sugar framework of the two section of DNA and bond the two sections together. Therefore, sticky ends allow for formation of recombinant DNA, if both were cut by the same restriction endonucleases.

After DNA is cut into fragments, it is necessary to find the fragment with the required gene which is done using a DNA probe. Then the fragment is cloned either by:

In vivo, by transferring the fragments to a host cell using a vector In vitro, using the polymerase chain reaction

To the DNA fragments, promoters - region of DNA to which RNA polymerase and transcription factors can attach to - are added. Also a terminator - a region of DNA that releases RNA polymerase and so ends transcription - is added.  

In vivo involves a vector to transfer DNA from one place to another, in this case a host cell. There are different types, but most common one used is the plasmid. If the same restriction endonucleases is used to break up the plasmid as well to cut up the gene, the DNA fragment will have complementary sticky ends to plasmid stick ends. This allows for the DNA fragment to be incorporated with the plasmid, forming recombinant DNA permanently using DNA ligase. This process is called insertion.

The following process is transformation, which is when recombinant DNA, in this case in form of a plasmid, is reintroduced into bacterial cells.

Bacterial cells and plasmids are mixed together in a medium containing calcium ions Calcium ions and changes in temperature, makes the bacterial membrane

permeable, allowing plasmids to pass through the cell-surface membrane into the cytoplasm.

Not all DNA fragments with the desired gene for the desired protein will be incorporated into the cells. Reasons include:

Only 1% of bacterial cells take up plasmids when the two mixed together Some plasmids will have closed up again without incorporating the DNA fragment

Sometimes the DNA fragment sticky ends join together to form its own plasmid

Following incorporation is identification of which host cells possess the desired gene. One of the older methods involved antibiotic resistance genes present in the plasmids. Some plasmids carry genes for resistance to more than one antibiotic. An example of this is the R-plasmid, which carries genes for resistance to two antibiotics - ampicillin and tetracycline.  To test which host cell incorporated the gene is done by the following process:

All the bacterial cells are grown on a medium that contains the antibiotic ampicillin. Bacterial cells that have taken up the plasmids will have acquired the gene for

ampicillin resistance These bacterial cells are able to break down ampicillin and so survive. Those who would taken up the plasmids would not have resistance to ampicillin and

so will die. This method is effecting in telling if bacteria have taken up the plasmids, but some bacterial cells would have taken up the gene without incorporating it as it closed and those were able to survive. To find out if the genes were incorporated, marker genes are used.

There are a number of different ways of using marker genes to identified whether a gene has been taken up by bacteria cells. They all involve using a second, separated gene on the plasmid. This second gene in identified for a reason, such as:

- Resistant to an antibiotic. This is an old technology. The DNA fragment is inserted into a place of one locus of a certain antibiotic-resistance gene and the other type remains functional. This allows to find which have taken up the plasmid (the intact one functioning) and if they have recombinant DNA (by seeing that the other is not functional/does not provide resistance). This process is called replica plating.

- Make a fluorescent protein that is easily identified/seen. This protein comes from jellyfish and green fluorescent protein (GFP). This process is rapid.

- Produce an enzyme who action can be identified. For example having a gene that produces the enzyme lactase. Lactase will turn a particular colourless substrate blue.

Summary:- Isolation of DNA using reverse transcriptase or restriction endonucleases- Insertion of DNA fragments into a vector e.g. plasmid, using DNA ligase. - Transformation – introduction of DNA fragment into suitable host cell- Identification of host cells that have taken up he DNA using gene markers- Growth/cloning – culturing of host cells containing the DNA to produce the protein on

a large scale

Polymerase chain reaction (PCR) – method of copying fragments of DNA. Is an in vitro method. It is an automated method that is rapid and efficient. It requires:

- A desired DNA fragment - DNA polymerase – the enzyme used is tag polymerase which is obtained from

bacteria in hot springs and is therefore tolerant to head (thermostable) and does denature during high temperatures during the process of PCR

- Primers – short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments

- Nucleotides - Thermocycler – a computer-controlled machine that varies temperature precisely

over a period of time

The process of PCR:1. Separation of the DNA strand . DNA fragments, primers and DNA polymerase are

placed in a vessel in the thermocycler. The temperature is increased to 95 degrees

Celsius. This causes the hydrogen bonds between two polynucleotide strands to break, separating them.

2. Addition ( annealing ) of the primers . The mixture is cooled to 55 degrees Celsius. This allows the primers to anneal to their complementary bases at the end of the DNA fragment. The primers provide the starting sequence for the DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separates strands from re-joining.

3. Synthesis of DNA . The temperature is increased to 72 degrees Celsius. This is the optimum temperature of the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.

The whole temperature cycle can take about 2 minutes. Over a million copies can be made in 25 cycles and 100 billion copies can be manufactured in just a few hours.

Advantages of in vitro cloning:- It is extremely rapid . This is especially useful when required information quickly, such

as in forensics. The only complication is that any contaminating DNA it would also multiply very quickly.

- It does not require living cells . No need for complex culturing techniques, time and effort.

Advantages of in vivo gene cloning:- Useful if wished to introduce a gene into another organism . After it is incorporated

into a plasmid, it can be transferred into other organisms. Gene therapy. - Involves almost no risk of contamination . Because it is cut by the same restriction

endonuclease, it can match the sticky ends of the plasmid. Contaminating DNA would not able to join the plasmid. In vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to a false result.

- It is very accurate . The DNA copies has a few, if any, errors. Mutations can occur but very rare. PCR used to produce about 20% of inaccurate DNA, though technology has improved. Any errors or contaminants in the sample would be coped in subsequent cycles.

- It cuts out specific genes . This makes it very precise procedure, and so does not copy the whole DNA.

- Produces transformed bacteria that can be used to produce large quantities of gene products. It can produce proteins for commercial and medical use.

DNA probe – a short, single stranded length of DNA that has some sort of label attached to it to make it easily identifiable. Two commonly used probes:

- Radioactively labelled probes – the nucleotides contain isotope 32P. The probe identified using an X-ray film.

- Fluorescently labelled probes – emit light (fluoresce) under certain condition e.g. when the probe has bound to target DNA sequence.

DNA probes are used to identify particular alleles of a particular gene by the following method. It can be used to find mutant allele that causes a particular genetic disorder or increase probability of certain diseases.

- The probe is made from a complementary base sequence to a section of the allele. This was original done using DNA sequencing techniques, however, now exists extensive genetic libraries for most genetic diseases.

- A fragment of DNA is produced complementary to mutant DNA. A marker is attached to it.

- Many copies of the probe are made using PCR.

- The double-stranded DNA that is being tested is treated to separate its two strands. This is done by denaturing DNA at high temperature.

- The strands are mixed with the probe and cooled. The probe binds to one of the DNA strands, DNA hybridisation. DNA also can recombine.

- Many different probes can be used to test for variety of different genetic disorders simultaneously.

- DNA is washed clean of any unattached probes. - The site at which the probe binds can be identified by the radioactivity or

fluorescence that probe emits.

Genetic screening can determine the probabilities of a couple having an offspring with a genetic disorder, especially when the allele is recessive. Potential parents can obtain advice from a genetic councillor about the implications of having children, based on their family history and the results of the genetic screening.

Genetic screening can be also used to detect oncogenes that are responsible for cancer. Some people inherit one mutated tumour suppressor gene, and so are at greater risk of developing cancer as if the other gene mutates, cell division will not be inhibited if it goes out of control and a cancer will develop. With genetic screening, individuals who contain the mutated gene can regulate their lifestyle (low mutagens) and treatment (check themselves more regularly to get an earlier diagnosis).

Another advantage of genetic screening is personalised medicine based on the individual’s genotype. Due to genes, some drugs may be more or less affected in treating a condition. This could save money, avoid harm or avoid raising false hopes. An example of these are painkillers, as to function effectively they need a specific enzyme to activate them. Another example is vitamin E, which for some individuals taking it can increase risk of cardiovascular disease and for other decrease.

Genetic counselling – special form of social work, where advice and information are given that enable people to make personal decision about themselves or their offspring.

Genetic fingerprinting – a diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis. It relies on the fact that most of eukaryotic DNA contains many introns. 95% of human DNA is made of introns, they are functional but yet still uncertain what they code for. DNA bases which are non-coding are known as variable number tandem repeats (VNTRs). Every individual has a unique number and length pattern of VNTRs, apart from identical twins. Two individuals have identical VTRs sequence is extremely small. The more closely related individuals the more similar are the VNTRs.

Gel electrophoresis – used to separate DNA fragments by their size. The DNA fragments are placed on to an agar gel and a voltage is applied across it. The fragments are attracted to the opposite side due to the voltage. The gel provides the resistance and so the longer the DNA fragment, the slower it would move and so in set unit of time would move less than a shorter fragment. This allows different lengths of DNA to be separated. If the DNA fragments are labelled, e.g. with radioactive probe, the position of the fragments can be known. Only DNA fragments up to around 500 bases long can be sequenced in this way. Larger genes and whole genomes must therefore be cut into smaller fragments by restriction endonuclease.

Origin – a point on the gel where the mixture of DNA fragments is applied.

Making of a genetic fingerprint:

1. Extraction of DNA from the rest of the cell. The amount is usually small and so its quantity is increased by PCR.

2. Digestion – DNA is cut into fragments using the same restriction endonucleases. They are chosen to cut close to, but not within the target DNA.

3. Separation of DNA fragments according to size by gel electrophoresis under the influence of an electrical voltage. The gel is then immersed in alkali in order to separate the double strands into single strands.

4. Hybridisation – radioactive or florescent DNA probes bind with VNTRs. The probes have a specific base sequence which is complementary to VNRTs and bind to them under specific conditions, such as temperature and pH. The process is carried out with different probes, which bind to different target DNA sequence.

5. Development – X-ray film is put over the nylon membrane, or the position is located visually if using fluorescent probes. A series of bars are revealed. The pattern of the bands is unique to very individual except identical twins.

To find if the sample belongs to certain individual, first the sample and the individual are visually checked. It there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of the DNA fragments from the bands. It does this using data obtained by measuring the distances travelled during electrophoresis. Then the odds of someone else having an identical fingerprint are calculated. This calculation is based on the assumption that DNA which prodcues the banding patterns is randomly distributes in the population. This may not be always the case, as for example, in religious and ethnic groups members of that group often have relations with each other and may not be the majority of the population. The closer the match between two patterns, the greater the probability that the two sets of DNA have come from the same person.

DNA fingerprinting has a variety of uses:- Genetic relationships – can be used to resolve questions of paternity. An individual

would inherit half of their genes from their mother and half from their father, so each band would either correspond to father’s or mother’s bands. If it does not, the child is not of that person/people.

- Genetic variability in a population – the more similarities between the bands, the more closely related individuals are. So if many individuals have similar bands, the population have low genetic variability; if less similar bands than high genetic variability.

- Forensic science – DNA is often left at the scene of the crime. Genetic fingerprinting can establish if an individual is likely to have been present at the crime scene, though they may not have necessarily performed the crime if the bands are a closed match. Other possible explanations need to be investigated. As well probability that someone’s else DNA might match that on the suspect has to be calculated. For example, other possible explanations might include:

DNA may have been left on some other, innocence occasion DNA may belong to a very close relative DNA sample may have contaminated after crime either by suspect’s DNA or by

chemicals that affected the action of the restriction endonucleases used in preparing the fingerprint.

- Medical diagnosis for diseases such as Huntington’s disease, which is a genetic disorder of the nervous system. It results from a three-base sequence (AGC) at one end of a gene on chromosome 4 being repeated greater than usual. People with fewer than 30 repeats are unlikely o get the disease, while those with more than 38 repeats are almost certain to do so. If they have over 50 repeats, the onset of the disease will occur earlier than average. A sample of DNA from a person with the

allele of the disease can be cut by restriction endonucleases and DNA fingerprint is prepared. This can then be matched with fingerprints of people with various forms of the disease and those without the diseases. The probability of developing the symptoms and when can be determined.

- Medical diagnosis to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with that of known pathogens.

- Plant and animal breeding – can prevent undesirable inbreeding during breeding programmes on farms or in zoos. Also, it can be used to identify which individuals have the desired gene. This allows individuals with that desired allele to be selected for breeding in order to increase the probability of their offspring having the characteristic that it produces. It also can be used to determine paternity in animals and establishing the pedigree of an individual.