猪白细胞Ⅱ类抗原基因多态性研究进展

本文对SLAⅡ类基因的染色体图谱定位、分子结构、基因分型及其多态性的研究进展作了介绍。重点介绍了SLAⅡ类基因的RFLP基因分型及其多态性研究。猪的主要组织相容性复合物Ⅱ类抗原基因座位存在不同程度的等位基因多态性。RFLP技术是一种简便而快捷的SLAⅡ类等位基因分型方法。SLAⅡ类基因的多态性和抗原肽结合位点密切相关,这些位点能结合处理过的抗原,然后递呈给T细胞。另外,在编码β1链的SLA-DRB1基因和SLA-DQB基因中有4个富含GC序列区。 Abstract:This article gave a detailed introduction about regional map,molecular structure,genotyping and polymorphism of SLA class Ⅱ genes.The pig major histocompatibility complex (MHC) class Ⅱ antigens have been known to exhibit a different degree of allelic polymorphism.The locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA class Ⅱ from genomic DNA.SLA class Ⅱ polymorphism was related to the antigenic peptide binding sites.Detailed analysis of sequences showed that there were 4 GC-rich sequences in exon 2 of SLA-DQB and SLA-DRB1 genes.     

Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles

The genetic diversity of the bovine class IIDRB3 locus was investigated by polymerase chain reaction (PCR) amplification and DNA sequencing of the first domain exon. Studying 34 animals of various cattle breeds, 14 previously unrecognized DRB3 alleles were identified. In three alleles, amino acid substitutions were observed that had not been previously found in bovine DRB3, but occurred at the same position in bovine DQB and in the DRB alleles of other mammals. For all newly identified alleles, the restriction fragment length polymorphism (RFLP) patterns of PCR products obtained with the enzymes Rsa I, Bst YI, and Hae III were compared with patterns of 38 previously described alleles. Altogether, eleven novel PCR-RFLP types were defined. Twelve out of the 42 PCR-RFLP types identified so far were not found to be fully informative because they corresponded to more than one allelic sequence. PCR-RFLP may therefore be a rapid and useful method for DRB3 typing in cattle families, but for studies on outbred populations, sequencing and hybridization techniques are required.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Microsatellite markers from a microdissected swine chromosome 6 genomic library

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.     

Canine Mhc DRB1 genotyping by PCR–RFLP analysis

A polymerase chain reaction (PCR) genotyping procedure has been developed for the canine major histocompatibility complex DRB1 gene ( Cafa-DRB1 ), which allows us to distinguish all the DRB1 alleles described to date and reveals the existence of new ones. The polymorphic second exon of the Cafa-DRB1 gene was amplified and the product analysed for restriction fragment length polymorphism (RFLP) with the enzymes Rsa I, Mbo I, Taq I and Asp HI. Nine RFLP combinations could be associated with previously known alleles. Two new RFLP combinations corresponded to new alleles and were confirmed by DNA sequencing ( Cafa-DRB1 *10 and Cafa-DRB1 *11). Close associations between RFLPs in the DRB1 second exon and the presence of specific amino acid residues included in the side-chain pockets of the antigen-binding site of the DR molecule are also described.     

Construction of Genetic Fingerprints of Aneurolepidium chinensis Using Microsatellite Sequences

The genetic fingerprints of Chinese wildrye ( Aneurolepidium chinensis (Trin.)Kitag) were constructed by Southern blot analysis of Ase Ⅰ-,Dra Ⅰ-,Eco RⅠ-,Hin dⅢ-,NcoⅠ-,MobⅠ-,RsaⅠ-,PstⅠ-, TaqⅠ- digested genomic DNA probed with synthesized oligonucleotide,（CT）8,（GCTA）4,（GGAT） 4 or（GACA） 4 .The difference between “Jisheng 4" and Chinese wildrye was shown by RFLP analysis of Dra Ⅰ-or Rsa Ⅰ- digested genomic DNAs probed with GCTA）4 .DNA fragments were obtained by PCR performed with genomic DNA of “Jisheng 4" as a template and（CT）8,（GCTA）4,（GGAT）4　or（GACA）4 as primer. Five size 2325,1455,876,774 and 299 bp fragments were amplified when (GGAT) 4 was used, indicating satisfied results were obtained.     

SNP genotyping on a genome-wide amplified DOP-PCR template

With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/µl improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.     

Detection of Genetic Variation in Alternaria brassicae by RAPD Fingerprints

Genetic variation in fourteen isolates of Alternaria brassicae collected from different geographical regions of the world was determined by RAPD (random amplified polymorphic DNA) analysis. Twenty random primers were tried to amplify genomic DNA of A. brassicae. Based on the PCR (polymerase chain reaction) amplification of genomic DNA of A. brassicae with four oligonucleotide random primers, fingerprints were generated for each isolate and the amplifed products were compared. Using this technique, intra- and intercontinental genetic variation among isolates of A. brassicae could be distinguished.     

Primase-based whole genome amplification

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37 degrees C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 10(8)-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

基于荧光定量PCR扩增反应的SNP测定法

建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.     

Parentage analysis for three Romanian families by DNA-fingerprinting

In forensic medicine, DNA fingerprinting for human identification and paternity testing is becoming a necessary procedure. The genetic locus D1S80 (MCT118) with Hinf I polymorphism of its 5' flanking sequence, HUMTH01 and D21S11 have been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels or high-resolution SDS-PAGE. Analysis of these VNTR loci was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permitted good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another. The aim of this study includes three paternity testing cases; they are the first three human DNA-fingerprints performed in Romania.     

Evaluation of Cryptosporidium parvum genotyping techniques.

We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.     

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.     

The conversion of RFLP markers to allele specific amplicons linked to QTLs governing malting quality in barley

Application of marker-assisted selection with RFLP based markers has been constrained by high cost and time requirements in situations involving a large number of plants. RFLP markers mapped on a Harrington/TR306 population have been identified elsewhere as linked to quantitative trait loci (QTL) governing malting quality. The probes ABG610, ABC622, as well as probes for the Nar1, Amy1 and Nar7 were sequenced and locus specific primers developed. These locus specific primers were applied to genomic DNA from both Harrington and TR306. Sequence analysis of the resultant monomorphic fragments revealed sequence divergence for the Xabg610, Xabc622, Amy1 and Nar1 loci, but not for the Nar7 locus. Application of a set of Hor2 primers to genomic DNA from the barley lines Harrington and TR306 led to the direct amplification of codominant alleles. Allele-specific primers were designed based on the sequence divergence identified among the Xabg610, Xabc622 and Nar1 alleles. Amplification conditions were optimized for each of these alleles such that only the favourable allele from Harrington was amplified. The usefulness of these primers for selecting Harrington alleles was demonstrated by their failure to amplify the corresponding alleles from the lines, Sterling, Stella and WM872. The Amy1 allele-specific amplicon was only capable of differentiating this locus between Harrington and TR306. The conversion of these markers into PCR amplifiable, allele-specific amplicons would greatly facilitate their application to barley breeding programs.     

A New PCR Method: One Primer Amplification of PCR-CTPP Products

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.     

Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli.

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.