Biology

Many DNA based diagnostic tests have been developed after the discovery of structure, function and duplication were discovered. Over the last 5 decades these techniques have undergone rapid changes and modifications to suit different applications such in phylogenetic relationships and evolutionary tree constructions of different taxa including all the varieties of living organisms. The techniques have been created for extracting gene sequences of known disease related and desirable traits in all organisms. These gene genes have suitable modified for their stable transfer into other taxa, integration, and later, detection of the same by many diagnostic techniques in the host recipient taxa. One of the most popular techniques has been based on the Polymerase Chain Reaction, a property of a DNA polymer formation by an heat tolerant enzyme called the Taq polymerase from thermo-stable Thermus aquaticus ( a fungus). In this paper this reaction has been used for the detection of a meat sample of an unknown organism. Its many applications have been briefly described. 
 
Identification of unknown meat samples has been very crucial in various kinds of situations such as food industry. In food industry meats of many organisms look alike with minor differences in texture, color, softness. Meat from cheaper and easily available organisms can be adulterated with processed food. Meat can also be contaminated with harmful pathogenic bacterial andfungal organisms andor also their toxins.

    Since the discovery of the molecular structure of DNA in 1953 by Watson and Crick and later a several researches on sequences, functions and expressions, many new areas of applications of DNA have become easier. No other diagnostic test has been found to be as real and true as the DNA based tests. DNA characterization tests have been of major value in evolutionary understanding and phylogenetic affinities of any organism. 

       Among the DNA tests many versions have been developed to achieve precision, economy of time and costs and minimizing the use of hazardous chemicals such as radioactive and highly toxic substances. In most of them, restrictions enzymes have been of crucial and key role. A restriction enzyme cuts the DNA at very enzyme specific sites. These DNA fragments are subjected to separation by electrophoretic methods on agar or agarose gels. The separated DNA fragments are probed with known radioactively labeled DNA sequences. These probes bind to the fragments which have these very sequences partially or fully. The technique has bee simplified considerable for diagnostics in shorter time by the polymerase chain reaction methods.

    A rapid test for meat from unknown animal sources has been described many times. Many tests have also been described and published. Buntzer et al (2005) used the satellite DNA markers as probes. Earlier Locksley and Beardsley (2000) described a DNA based method for authentication of the source. This has many applications in the food industry 

In this paper a molecular analysis experiment was done to characterize a meat tissue of unknown animal origin. DNA was extracted and mitochondria specific DNA (mtDNA) fragments were amplified by polymerase chain reaction. The positively amplified sequences were then cycle sequenced using a non-radioactive fluorescent dye. The DNA sequences so obtained were found to be 293 base pairs. These sequences were then compared with the large vertebrate DNA sequence Data Base on computers. The comparison provided the most probable identity of the organisms involved.

Materials and Methods
DNA Extraction  DNA from the unknown sample meat tissue supplied was extracted using the Qiagen animal DNA extraction kit ( 69506). The meat sample tissue was thawed in a water bath set to 55 0 C.  The tissue held by a pair of sterile was cut into A small piece (about 25 mg) was tissue was cut out by a pair of sterile scissors  from the thawed tissue held by a pair of sterile forceps. This piece was dispensed into a 1.5 ml tube labeled with the supplied meat tissue sample no. on the lid of the tube by a sterile tooth pick. Into this sample 180 ul of ATL buffer using a sterile tip on a blue pippettor. The tissue was lysed using 20 ug20 mg per ml Proteinase K using a yellow pippettor and mixed thoroughly. This tube was then incubated at 55 0 C for 45 minutes while shaking.

A water bath was heated to reach 70 0 C and allowed to remain at that temperature. The lid of the sterile DNAeasy spin column was labeled with the provided meat sample number. This was into a sterile 2 ml collection tube. Another 1.5 ml sterile tube was labeled with the sample and T DNA (Total DNA). After thorough mixing the sample, the lysis was stopped using 200 ul AL buffer. After mixing this 200 ul of 100 ethanol was added and mixed again. This mixture was transferred with a pippettor into the spin column in the 2 ml labeled tube. This was centrifuged at 8,000 rpm.  After slowly decanting the supernatant, the spin column was put back into the collection tube. The precipitate was washed with 500 ul AW1 buffer and spun again at 8,000 rpm. The spin column was put again into collection tube washed again with AW2 buffer and then spun at 11,000 rpm for 3 minutes. After decanting the supernatant the DNA easy spin column was taken again into 1. 5 ml tube. To elute 200 ul AE buffer was added to the membrane to the spin column. This was left for 1 minute at room temperature. This was spun at 8,000 rpm and then the spin column was removed into dust bin leaving DNA in the collection tube. This DNA sample was then electrophoresed to determine the success of the above procedure.
Polymerase Chain reaction.- To each tube 47 ul of supplied Master Mix (containing 35.5 ul ddH2O, 10 ul of salt buffer including Mg Cl2, 10 ul of dNTPs, 0.5 ul of Taq polymerase) with separate new pippette tips. The to each the following were added using a new and separate pippette tips
1 ul of forward primer (NM)
1 ul of reverse primer (PIL)
1 ul of our test DNA as prepared above

        The lids were tightly closed and put on a strip tube each.
    Thermal cycling was conducted as guided with a GENE AMPR PCR System 9700. This goes through40 cycles of 1 min each (94 0 C for 3 minutes for denaturation1 min at 54 0 C for primer annealing and 4 min at 72 0 C for primer extension) after initial incubation at 94 0 C for 3 minutes. There was a final step of 10 minutes at 72 0 C for termination of the Polymerase Chain Reaction (PCR).
    This DNA sample was electrophoresed on 1 agarose gels. These gels were stained with ethidium bromide to reveal the ethidium bromide stained bands under UV light.
Cycle Sequencing Reaction
    Quantity of clean up PCR product was determined by comparing the intensity of ethidium stained bands of the test meat DNA sample with that of the PGEM band. If the intensity was equal 2 ul of the test meat DNA sample was used. In our sample preparation intensity was more than the intensity of the Ethidium bromide staining PGEM band. Therefore 1 ul of the sample was used.
To the 0.2 ul tube labeled as meat sample number, the following solutions were added
19 ul of dd H2O
1 ul of Primer ND4
1 ul of PCR test sample DNA product
3 ul of Quick Start DTCS (Direct Terminator Sequence mix) from Beckman Coulter Inc.)
The tubes were tightly closed for thermal cycling reaction. Gene Amp PCR System 9700 was used for thermal cycling. This goes through 40 cycles  20 seconds at 96 0 C for denaturation, 20 seconds at 50 0 C for primer annealing and 4 minutes at 60 0 C for primer extension.

Cycle Sequencing Reaction Clean up 
    The meat test sample DNA obtained as above is once again is cleaned. To the tube 5. 0 ul of Prep solution (2. 0 ul 100mM EDATA, 2.0 ul of 3 M NaOAc, 1. 0 ul of glycogen) is added. To this 60 ul of cold 95 ethanol is added. This was mixed thoroughly and centrifuged at 13, 200 rpm for 15 minutes.  The supernatant was removed by a pipette without disturbing the pellet.  Then 200 ul of cold 70 ethanol was added, mixed thoroughly, spun again the supernatant was once again removed. Cold ethanol 70 was added again and centrifuged at 13, 200 rpm for 5 minutes. The sample was air dried for about 30 minutes at room temperature till all the ethanol was evaporated. 
Electropherograms, Sequence Analysis
    The success of the above reactions was assessed by electrophoresis in 6
Polyacrylamide gels in the Beckman Coulter CEQ 8800 DNA Sequencer. This contains the LASER which reads the signals from the fluorescently labeled ddNTPs    
The DNA sequence used for comparing the 16 base pair ATCGATCTACGATCG sequence.
The sequence of the test meat sample as obtained in the Beckman Coulter Sequencer was compared with the publicly available annotated  sequences 13 billion bases from over 100,000 available at the NCBI ( National Center for Biotechnology Information of the National Library of Medicine - National Institutes of Health, USA  web site). This Gene Bank of this Center contains other world wide updated information on Gene Bank,  Blast, Taxonomy and published research papers from a large number of world wide sources.
Basic Local Alignment Search Tool (BLAST) is used for carrying out the alignment and similarity index analysis.
Results

     Table 1 gives the list of animals with Similarity Index over 75. Maximum ID observed was in the range of 75  81. The logical choice of the animal closest to the test meat sample may most probably be Drymarchon corais which showed the maximum similarity of 81.

Discussion
One of the NCBI responsibilities is to facilitate the easy methods for data searches in addition to creating vast data bases on molecular biology information from all the published and available real data and storing them in manageable manner.   In searching for similarity information a parameter of low E value is chosen. In our experiment an E value fixed was 0.0  e 0.154  6e 0.21  1e 0.13  5e 0.07.
Nine plant species were found to have the range of high similarity index between 74 - 81. Among them Drymarchon corais was found to be the closest with 81 similarity to the meat test sample.
Many varieties of DNA based tests have been described and published. All these publications have facilitated the use of DNA based tests for various purposes such as the authentication of the source of meat Bunter et al., 2005 identification of meats from different samples (Locksley and Beardsley, 2000), evaluation of pathogenic Data et al.,  for Hemolytic  Listeria monocytogenes) and infectious organisms (Ellingson et al., Salmonella Dragan and Avraham, 2000 Salmonella in poultry, Fluit et al., 1993) and or their toxins causing serious health hazards to consuming humans or cattle and poultry (by colony hybridization, Datta et al 1987).
The attraction of the newly developing DNA technology for detection of large numbers of sample in short time was reported as 50 samples in just 4 hours (Buntzer et al 1995). Polymerase Chain reaction was used for pork in heated in meat products (Meyer et al., 1994)