Simple and rapid human papillomavirus genotyping method by restriction fragment length polymorphism analysis with two restriction enzymes
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Abstract Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low‐cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes Hpy CH4V and Nla III. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR‐positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non‐specific amplifications with PCR. In conclusion, this PCR‐RFLP method using restriction enzymes Hpy CH4V and Nla III is simple, non‐labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples. J. Med. Virol. 85:1229–1234, 2013 . © 2013 Wiley Periodicals, Inc.Keywords:
Papillomaviridae
We have explored the role of the kallikrein—kinin system in essential hypertension using spontaneously hypertensive rats (SHR) as an animal model. A rat tissue kallikrein complementary (c) DNA (RSK 1105) was used as a probe in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs) in SHR. Using 23 different restriction endonucleases, we have identified five RFLPs involving alterations in restriction fragment lengths for the restriction enzymes Bgl II, Dra I, Nde I, Sph I, and Bcl l. Three of the enzymes, Nde I, Sph I, and Bgl II, generate multiple polymorphic fragments. We have further mapped these RFLPs with two additional probes, both from the rat renal kallikrein gene RSKG 7. The 5' probe, consisting of sequences approximately 2000 base pair (bp) 5' of the first exon, recognizes RFLPs in DNA digested with Bel I and Sph I. The 3' probe, approximately 4400 bp away from the fifth exon, recognizes polymorphic fragments in DNA digested with Bel I, Dra I and Nde I. These findings indicate possible differences in tissue kallikrein genes or their regulatory regions in SHR that could contribute to the pathogenesis of hypertension in this animal model.
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Research about PCR-RFLP gene 16S rRNA in toad (Bufonidae) using restriction ezymes has conducted from October 2016 to January 2017. The aim of this research is to detect variation from three species of Bufonidae using PCR-RFLP method gene 16S rRNA. DNA sampel was isolated dan amplified by PCR process. RFLP was done using two restriction enzymes, MseI (Tru1I) and HphI. For restriction enzyme MseI produce different restriction sites and fragment that are vary in length for three species of toad that makes us able to detect variation. RFLP with restriction enzymes HphI produce restriction sites and fragments that are equal in length so it can’t be use to detect variation between three species of toad from genus Duttaphrynus and Phrynoidis
Keywords: Bufonidae, PCR-RFLP, 16S rRNA, primer, restriction enzyme
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This investigation was undertaken with the objective to study DGAT1 gene exon 8 polymorphism using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in 53 Surti and 56 Banni buffaloes. The restriction digestion of 412 bp product with AluI revealed only one genotype AA in both Surti and Banni buffaloes. The frequencies of allele A were observed as 0.55 and 0.54 in Surti and Banni buffaloes, respectively on restriction digestion with HincII. The restriction digestion of amplified product with HphI revealed two fragments. The frequency of allele A were 0.71 and 0.36 in Surti and Banni buffaloes, respectively. We found that the 412 bp DGAT1 gene fragment was fairly polymorphic with HincII and HphI restriction enzymes, while monomorphic with AluI restriction enzyme in both buffalo populations studied.
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In the present study, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was developed to identify 5 species ( Trichinella spiralis , Trichinella britovi , Trichinella nativa , Trichinella nelsoni and Trichinella pseudospiralis ) and 3 phenotypes of uncertain taxonomic status ( Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory–secretory (E–S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the Hinf I or Dde I endonuclease restriction of the 2800 bp fragment; T. nativa by the Rsa I restriction of the 2800 bp fragment, or by the Alu I restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the Alu I restriction of the 1250 bp fragments, and can be discriminated between them by the Ssp I restriction of the 2800 bp fragment; T. pseudospiralis by the Msp I restriction of the 372 bp fragment; T. nelsoni by the Hha I or Alu I restriction of the 2800 bp fragment; Trichinella T5 by the Hha I restriction of the 2800 bp fragment; Trichinella T6 by the Alu I restriction of the 1250 bp fragment; and Trichinella T8 by the Ssp I or Rsa I restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi , Trichinella T5 and T6.
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Trichinella spiralis
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A restriction fragment length polymorphism (RFLP) is defined by an enzyme, a restriction endonuclease, that cuts the doublestranded DNA at a particular sequence of bases, a probe, a labeled, complementary segment of DNA that will anneal to a portion of the digested sample, and a set of variable fragment length bands that appear on a Southern blot. An elementary discussion of restriction enzymes, their nomenclature, recognition sequences, and activities is presented. This is followed by a basic description of the RFLP and its components, electrophoresis, the Southern blot, and hybridization. Simple DNA polymorphisms are illustrated in light of the nomenclature for RFLPs in human gene segments of known function and those that are anonymous. It is shown that the majority of RFLP loci have two alleles, while a subset, the minisatellite and some variable number of tandem repeat (VNTR) loci, exhibit hypervariability. Applications of RFLP technology to parentage testing, human gene mapping, prenatal diagnosis, and evolutionary studies complete the essay.
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The restriction endonuclease maps of mitochondria DNA (mt DNA) from the eggs of 34 domestic silkworm ( Bombyx mori) strains were analyzed through 29 different restriction endonucleases.mtDNA of multivoltine strains showed different RFLP (restriction endonuclease length polymorphism) from that of monovoltine and bivoltine strains,but no RFLP was shown to exist between different geographical strains.RFLP was also observed between fertilized and unfertilized eggs of the same breed in their mtDNA.Restriction endonuclease maps with moderate preciseness were constructed of domestic silkworm.
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Thirty four taxa of Actinidia were investigated through PCR RFLP analysis of two mitochondrial DNA fragments, including nad1/B C gene and nad4/1 2 gene. Digestion of these two fragments by seven restriction endonucleases yielded twenty polymorphic restriction fragments. We only found restriction fragment length variation caused by insertion or deletion. It suggests that mtDNA restriction length polymorphism could be an efficient indicator for conservation biology .
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Summary IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.
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BstⅪ and HhaⅠ polymorphism of pGH gene's full sequence by PCR-RFLP in Yorkshire and Landrace is analyzed. The results of RFLPs analysis indicate that there is only one BstⅪ restriction enzyme site, without discovering BstⅪ restriction enzyme site polymorphism in the pGH gene full length, but there are abundant HhaⅠ restriction enzyme site polymorphisms. After digesting with HhaⅠ restriction enzyme, the amplified 2107?bp DNA segments show 5～9 restriction enzyme sites and 6～10 fragments. A～J ten band-types in Yorkshire pigs and eight band-types in Landrace pigs can be distinguished, and F and I band-types in Landrace pigs are not discovered. The frequency of B band-type is the highest; it is 31.11% in Yorkshire and 41.67% in Landrace, the difference is the most significant (P0.01) between Yorkshire and Landrace. However, the distribution differences of other band-types are not significant (P0.05) between two breeds pigs.
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Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to differentiate between serotypes of several infectious bronchitis virus (IBV) strains. A sequence of 1720 base pairs (bp) that contains the S1 glycoprotein gene of IBV was amplified by PCR, purified, and digested with restriction enzymes. Eleven reference IBV strains were grouped according to the RFLP patterns. The IBV Holte, Arkansas DPI, SE 17, Md 27, and Iowa 97 strains could be differentiated from the other IBV strains using the restriction enzyme HaeIII. The Beaudette, Massachusetts 41, Connecticut, and Florida 88 strains had the same HaeIII RFLP pattern but could be differentiated using XcmI and BstYI restriction enzymes. The Gray and JMK strains could not be differentiated by their RFLP patterns following digestion with 23 different restriction enzymes. Twenty-six samples (field isolates and reference strains) of IBV, previously serotypes by the virus-neutralization (VN) test in embryonating eggs, were analyzed in a blind fashion. The results using the PCR and RFLP analysis agreed with the serotype for traditional and variant IBV viruses as determined by the VN test.
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