Case Study of Genetic Analysis for Detection of Pork Mixed in Beef Jerky
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본 실험에서는 식품의약품안전평가원의 식품 중 사용원료 진위 판별 지침서에 명시 되어있는 소와 돼지에 대한 primer를 이용한 PCR 반응의 민감도와, 새롭게 선별한 primer의 PCR 민감도를 비교하였다. 새로운 primer의 타겟 부위는 cytochrome b이다. Primer의 적합성을 확인하기 위해 돼지고기와 소고기를 이용하여 DNA를 추출 후 PCR을 통하여 확인 하였다. 육포시료에서 식품의약품안전평가원의 식품 중 사용원료 진위 판별 지침서에 명시 되어있는 소에 대한 primer를 사용하였을 경우, PCR 산물을 얻을 수 있었음에 반해, 돼지 primer를 이용하였을 때는 PCR 산물을 얻을 수 없었다. 반면에 새로설정 한 primer에서는 전체 27개의 육포시료 중 13개에서 돼지 primer를 이용한 PCR 산물을 얻을 수 있었다. 이렇게 얻은 PCR 산물을 시퀀싱을 통하여 확인 해본 결과, 돼지 (Sus scrofa)임을 확인 할 수 있었다. 그러므로 본 실험에 사용 한 육포시료 중 일부는 돼지와 소고기의 혼합물임을 확인 할 수 있었다.We compared the sensitivity of polymerase chain reaction (PCR) using newly discovered bovine and porcine primers to previously reported primers of the Korea Food and Drug Administration (KFDA). The target site for the new primers was cytochrome b. The suitability of the primers was confirmed by conducting PCR on DNA extracted from beef and pork control. When the beef jerky samples were tested by PCR with the conventional primers of the KFDA, the DNA was successfully amplified from all samples with the bovine primer, whereas no PCR products were obtained with the porcine primer. By contrast, in the experiments with the new primers, we obtained PCR products from 13 of 27 beef jerky samples using the porcine primer. We have confirmed that the base sequences of these 13 samples match to the sequence of pigs (Sus scrofa) in the National Center for Biological Information (NCBI) database. Therefore, we determined that some of the beef jerky samples we received are mixture of beef and pork.Keywords:
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Abstract There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele‐specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR‐amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer–target complexes by using allele‐specific primer probes. PCR amplification by a DNA polymerase from matched 3′‐primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real‐time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4′‐ C ‐modified nucleotide residues at their 3′ termini, and their evaluation in real‐time asPCR. We found that primer probes bearing a 4′‐ C ‐methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.
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We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR products where minimal sequence information is known or as an alternative to primer walking when sequencing long PCR products. A cassette containing the hybridization site for the M13 sequencing primer is ligated to a sample PCR product. Using one phosphorylated primer specific for the cassette together with one primer specific for the sample PCR product, subsequent PCR amplifies one hybrid construct directionally. This allows utilization of the universal M13 primer when sequencing of one strand after the removal of the complementary strand using lambda-exonuclease.
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INTRODUCTION The objective of a polymerase chain reaction (PCR) is to amplify a specific DNA segment without any nonspecific by-products. In principle, each physical and chemical component of PCR can be modified to produce a potential increase in yield, specificity, or sensitivity. Yet the most critical parameter for successful PCR is optimal primer design. A poorly designed primer can result in little or no product, due to nonspecific amplification and/or primer-dimer formation leading to reaction failure, even when all the other parameters are properly optimized. This article provides general guidelines for PCR primer design, tips for development of primer pairs for more complex applications, and advice on the development of probes for real-time PCR.
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Abstract For PCR‐based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium , and species‐specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species‐specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species‐specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.
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Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single- and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.
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The hiTAIL-PCR is an effective method completely based on PCR technique.A long arbitrary degenerate(LAD) primer with a higher degree of degeneracy is used to create primer-binding sites efficiently along the unknown target sequences.An additional sequence LAD0 identical to the 5' part of the LAD primer is utilized,resulting in primer concentration that is high enough to bind the unknown flanking sequence.Using the nested PCR is intended to improve specificity of PCR products.We firstly established the hiTAIL-PCR system to isolate the unknown target sequences in onion.
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A two-step polymerase chain reaction (PCR), with four double (nested) primer pairs, used for the detection of HIV-2 in clinical samples is described. With these four nested primer pairs we could detect HIV-2 DNA in 17 of 17 virus isolates and in blood mononuclear cell samples from 31 of 37 (83.7%) seropositive individuals after ethidium bromide staining of the amplified DNA. The nested primer PCR was also compared with a single primer pair-based PCR followed by hybridization. The sensitivities of the two methods were almost equal, but the nested primer PCR offered obvious technical advantages.
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A PCR approach was developed using a hexameric degenerate primer, which reflects the Shine-Dalgarno sequence of prokaryotic transcripts, hitherto named SD-PCR. In standard PCR reactions, the sizes and melting temperatures of the two primers are usually designed to be as equal as possible, while SD-PCR uses a single long gene-specific primer pairing with a much-shorter universal degenerate primer. This approach can be used in PCR walking to clone either the upstream or the downstream region of a known sequence. We have successfully applied the method to template DNAs of different GC contents as well as complex mixtures composed of highly contaminating DNA(s).
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clone (Java method)
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