LABORATORY
PCR TECHNIQUE
PCR is done in a single tube with appropriate chemicals and a specially designed heater . The technique was developed in 1983 by Kary Mullis and Michael Smith , which resulted in them receiving a Nobel Prize 1993 .
PCR utilises the following :
• Sample containing a nucleotide sequence ( from blood , hair , pus , skin scraping , etc .).
• DNA primers , which are short single , stranded DNA that attaches to nucleotide sequences that promotes synthesis of a complementary strand of nucleotides .
• DNA polymerase , which is an enzyme that , when the DNA has a primer bound , goes down the DNA segment attaching DNA building blocks to form complementary base pairs and thus synthesizes a complementary nucleotide strand of DNA .
A large excess of DNA building blocks termed nucleotides ( adenine , thymidine , cytosine and guanine ,) are present in solution . When these blocks are linked together , they form a nucleotide sequence or a single strand of DNA . In the solution , the single strand of DNA building blocks bind their complementary building block by weak hydrogen bonds ( for example , A will only bond with T and G only with C ) a complementary DNA nucleotide sequence is formed and bound to the original single stranded DNA . When the binding is completed , a complementary double strand DNA is formed in a specific sequence .
PCR begins with a segment of DNA from a sample that is placed in a tube with the reagents listed above . The solution is heated to at least 94 ° C ; this heat breaks the hydrogen bonds that allow complementary DNA strands to form , so only single strands exist in the mixture , which is termed denaturation of double stranded DNA .
The mixture is allowed to cool to about 54 ° C . At this temperature , the DNA primers and DNA polymerase bind to individual single stranded DNA ( termed DNA annealing ). Because the building blocks are in a high concentration in the mixture , the polymerase uses them to make new complementary strands of DNA ( termed DNA extension ). This process creates a new double-stranded DNA molecule from each of the single strands of the original molecule .
This cycle is repeated approximately 40 times in thermal cycler , which automatically repeats the heating-cooling cycles , with the amount of each DNA sequence doubling each time the heating-cooling cycle is completed . Thus a single short segment of DNA can be amplified to about 100 billion copies after 40 doubling cycles .
TYPES OF PCR
Multiplex PCR Multiplex PCR uses multiple , unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different segments of the DNA sequences . An important requisite for multiplex PCR is that amplicons generated must have sufficient size differences so that their base pair length can easily be differentiated when visualised by gel electrophoresis . The main use of this technique is in the simultaneous detection of numerous organisms in a single sample .
Nested PCR Nested PCR increases the specificity of DNA amplification by reducing background due to nonspecific amplification of DNA . Two sets of primers are used in two successive PCR reactions . In the first reaction , one pair of primers is used to generate DNA products , which , in addition to the intended target , may consist of non-specifically amplified DNA fragments . These product ( s ) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from each of the primers used in the first reaction . Nested PCR is often more successful in specifically amplifying longer DNA fragments than in conventional PCR , but it requires more detailed knowledge of the target sequences . For example , this technique has been used in the identification of Tritrichomonas foetus infections in cats .
Quantitative real-time PCR This is used to simultaneously amplify and quantify a targeted DNA molecule . It enables both detection and quantification of a specific sequence in a DNA sample . Real-time PCR is a technique growing in popularity in veterinary medicine . Its uses include the blood detection of Babesia species , Leishmania , and the simultaneous detection of multiple faecal helminth infections .
Canine Genetic Disease Testing Over the past few years , major advances have been made in the understanding of the molecular basis for inherited diseases in dogs . Most of the tests , available today have been developed using a comparative medicine approach to canine genetics , which utilises previously identified human diseases similar to the canine ones and tests the gene or genes responsible for the human disease to see if they are also mutated in the canine disease . Another approach is to identify genes that cause diseases in dogs , which have not previously been implicated in the equivalent human diseases . The latter approach is based that human populations tend to be more outbred than purebred dogs , which makes genetic analysis easier in dogs then in people .
There are two different types of tests available for DNA genetic testing , namely the direct and the indirect DNA test . Each of these different types of tests has their own sources of error and therefore has different implications for breeding decisions . Both of these types of testing could be used for clinical diagnosis of diseases when normal diagnostic tools might be invasive and carry with them life-threatening complications . Presently DNA based genetic tests are available for over 50 inherited diseases in dogs and the number of tests increases all the time . vet360
Issue 02 | APRIL 2017 | 22