Identification of BoLA-DRB3.2 alleles in Korean native cattle (Hanwoo) and Holstein populations using a next generation sequencer

Bovine leucocyte antigen (encoded by BoLA) has been widely studied to identify the association with many traits related to immunity. Exon2 of BoLA-DRB3 is extremely polymorphic, and more than 100 alleles have been identified. We investigated polymorphisms of BoLA-DRB3.2 in Korean native cattle and Holstein populations using a next generation sequencer of the GS-FLX Titanium system. We found 38 alleles including 11 new alleles (BoLA-DRB3*1303, *4702, *7101, *7501, *7201, *7301, *7601, *1104, *7701, *7401 and *50021) in Hanwoo, and nine alleles including one new allele (BoLA-DRB3*7601) in Holstein. The 454 sequencing method is a promising alternative technology for high throughput genotyping of BoLA-DRB3.2 because of its technical advantages that allow it to overcome the disadvantages of sequence-based typing methods.     

Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA-DRB3 alleles

The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3 -typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10–15% acrylamide combined with a 15–50% urea-formamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3 -typing.     

Identification and characterization of new BoLA-DRB3 alleles by heteroduplex analysis and direct sequencing

A sample of 52 mixed-breed dairy cattle (Holstein Friesian and Jersey) and 51 beef cattle (Hereford) from south-east Queensland was studied. The second exon of BoLA-DRB3 was amplified by polymerase chain reaction (PCR), and polymorphisms were detected by heteroduplex analysis. A large number of different heteroduplex patterns indicated extensive sequence polymorphism. Direct sequencing of PCR products from 17 homozygotes and cloning and sequencing of PCR product from two heterozygotes resulted in the identification and characterization of four novel alleles. The previously described allele BoLA-DRB3 * 2A is characterized by an amino acid deletion at position 65. We have identified three animals that are homozygous for this amino acid deletion, indicating that the deletion is unlikely to result in loss of function. Two of these animals had allele BoLA-DRB3 * 2A, and one had a novel allele with codon 65 deleted but differing from BoLA-DRB3 * 2A at a number of other amino acid positions. In conclusion, heteroduplex analysis allows rapid discrimination between homozygotes and heterozygotes, and enables rapid identification of new BoLA-DRB3 alleles.     

Analysis and frequency of bovine lymphocyte antigen (BoLA-DRB3) alleles in Iranian Holstein cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.     

Detection of a common BOLA-DRB3 deletion by sequence-specific oligonucleotide typing

The bovine MHC class II BoLA-DRB3*2A has an amino acid deletion of unknown function at codon 65 in the second exon, which codes for the antigen-binding site. Sequence-specific oligonucleotides were designed based on published nucleotide sequences on BoLA-DRB3 alleles, and used to detect this deletion in 51 Hereford cattle. Probes 65+ and 65- detect the presence or absence of codon 65 respectively. Oligonucleotide probes were labelled with Digoxigenin (DIG), hybridized to dot blots of BOLA-DRB3 exon 2 polymerase chain reaction (PCR) product, and detected by chemiluminescence. Of the 51 animals screened, two were homozygous and 11 were heterozygous for the deletion at codon 65. The methodology described here provides the necessary tools to screen rapidly for this deletion in a large number of animals in order to study its effect on antigen binding and immune response.     

Analysis and Frequency of Bovine Lymphocyte Antigen (BoLA-DRB3) Alleles in Iranian Holstein Cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11, and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2, and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4 and 4.4%, respectively. Thus, in the Iranian Holstein cows studied alleles associated with resistance to various diseases are found. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.__________From Genetika, Vol. 41, No. 6, 2005, pp. 817–822.Original English Text Copyright © 2005 by Nassiry, Eftekhar Shahroodi, Mosafer, Mohammadi, Manshad, Ghazanfari, Mohammad Abadi, Sulimova.The article was submitted by the authors in English.     

Variation of the BoLA-DRB3 gene in dairy cattle and its effect on the viability parameters

Genotyping of the BoLA-DRB3 alleles was performed in dairy cattle of Krasnodar krai and Holstein stud bulls. Loss of heterozygosity, which decreased the reproductive parameters, was observed. It was proposed that stud bulls be selected on the basis of their genotyping at the BoLA-DRB3 gene to prevent further decay of cattle viability.     

Extensive polymorphism of the BOLA-DRB3 gene distinguished by PCR-RFLP

A polymerase chain reaction (PCR)-based method is described for typing of alleles of the bovine lymphocyte antigen (BoLA)-DRB3 gene. A total of 30 DRB3 alleles were distinguished by digestion of PCR amplification products of BoLA-DRB3 exon 2 with RsaI, BstYI and HaeIII (PCR-RFLP). All restriction fragment patterns, with the exception of one HaeIII pattern, were consistent with restriction sites that were found among 14 previously sequenced DRB3 alleles. The PCR-RFLP typing method was evaluated on 168 genomic DNA samples collected from animals of 10 cattle breeds, 48 of which were typed in the Fourth International BoLA Workshop for BoLA-DRB and -DQ by conventional restriction fragment length polymorphism (RFLP) analysis using heterologous and homologous DNA probes. Thirty-one DRB/DQ haplotypes containing 23 DRB3 alleles were identified among the 48 workshop animals analysed. Using PCR-RFLP, 11 DRB3 alleles were identified in 18 workshop animals for which DRB RFLPs were not informative. PCR-RFLP typing of additional animals revealed five new DRB3 alleles, of which three contained a putatively located three basepair deletion in the identical position as found for the sequenced allele DRB*2A. PCR-RFLP was shown to be a rapid and sensitive method for the detection of polymorphism in a functionally relevant domain of the BoLA-DRB3 gene and should be useful for studying the evolution of DRB polymorphism in cattle and other Bovidae.     

BoLA-DRB3 gene polymorphism and FMD resistance or susceptibility in Wanbei cattle

For the further characterization of foot-and-mouth disease virus (FMDV)-induced foot-and-mouth disease, we investigated the association between polymorphism of BoLA-DRB3 gene and FMD resistance/susceptibility of Wanbei cattle challenged with FMDV. One hundred cattle were challenged with FMDV and exon 2 of BoLA-DRB3 genes was amplified by hemi-nested polymerase chain reaction from asymptomatic animals and from animals with FMD. PCR products were characterized by the RFLP technique using restriction enzymes Hae III. The results revealed extensive polymorphisms, 6 RFLP patterns were identified. By analyzing alleles and genotypic frequencies between healthy and infection with FMD cattle, we found that allele Hae III A was associated with susceptibility to FMD in Wanbei cattle (P < 0.05), whereas Hae III C was associated with resistance to FMD (P < 0.01) and may have a strong protective effect against FMD. Hae IIICC and Hae III BC genotype were associated with resistance to FMD (P < 0.01). By contrast, Hae III AA genotype was associated with susceptibility to FMD (P < 0.01). Sequence analysis show that 89 amino acids were translated in exon 2 of BoLA-DRB3 and 13.70 % of nucleotide mutated, which resulted in 14.61 % of amino acid change. One PKC, one Tyr and one CAMP phosphorylation were increased; the hydrophobicity and secondary structure of proteins produced change after amino acid substitution. These results revealed that Wanbei cattle had the ability of resistance to disease by mutation which result changes of the protein structure to perform the regulation of the cell using different signaling pathways in the long process of choice evolution.     

Comparative analysis of Ayrshire and Black Pied cattle breeds by histocompatibility markers

Distribution of BoLA-A antigens and BoLA-DRB3 alleles was studied by means of the microlymphocytotoxic test (BoLA-A) and the PCR-RFLP method (BoLA-DRB3) using restriction endonucleases RSAI, HaeIII, and XhoII in Ayrshire (n = 127) and Black Pied (n = 129) cattle breeds. Comparative analysis of profiles for class I antigens revealed significant differences in the frequencies of antigens W2, W6, W10, W31, W44, W15, and W19 (P > 99%). The studied breeds also differ in the spectrum of BoLA-DRB3 alleles and distribution of their frequencies. Heterogeneous allele frequency profile was detected in Ayrshire cattle: five of 18 detected alleles (DRB3.2*7, *8, *10, *24, and *28) accounted for 77%. Allele DRB3.2*7 (37.6%), which is classed with rare alleles in Black Pied cattle is the most common in Ayrshire cattle. The observed heterozygosity level in the combined sample of Black Pied breed (0.836) is higher than in Ayrshire breed (0.070). In both breeds, the heterozygosity level was studied in the groups of healthy and ill with persistent lymphocytosis (caused by bovine leukemia virus) animals and in the group of virus carriers in Ayrshire breed. In ill animals, a decrease in the observed heterozygosity level was detected, as compared to healthy animals and the expected heterozygosity level. The observed heterozygosity level exceeds the expected one in virus carriers. The detected features of the heterozygosity level in the studied groups allow the heterozygosity level for locus BoLA-DRB3 to be considered a nonspecific factor of resistance to leukemia and are heterozygous animals to have higher resistance to bovine leukemia. The presence of a larger proportion of highly productive animals (the annual productivity of more than 7000 kg) in the group of ill Ayrshire cattle animals, as compared to healthy animals to established. To increase resistance to bovine leukemia, the obtained data indicate the importance of the control of heterozygosity level and genetic diversity for gene BoLA-DRB3 in cattle herds.     

黄麂Mhc-DRB 基因多态性及其维持机制

利用牛DRB3 特异性引物(LA31 和LA32),通过聚合酶链式反应(PCR)、单链构象多态性(SSCP)以及克隆测序技术,从12 只黄麂个体中共获得20 个DRB 第二外显子等位基因,其中6 个个体具有3 ～ 4 个等位基因,提示利用该引物从黄麂中至少可以扩增出2 个DRB 位点。所有序列均无插入、缺失和终止密码子。基于序列比对(与牛DRB3 和鹿科DRB 基因同源性非常高),以及所检测到的氨基酸变异位点主要位于抗原结合区,推测本文所获得的黄麂序列为表达的、且具有重要功能的DRB 位点。抗原结合区氨基酸位点的非同义替换(d_N )显著大于同义替换(d_S )(P < 0.01),说明历史上黄麂DRB 基因经历过正选择作用。CODMEL 程序中的模型M7 和M8 似然比检测(Likelihood ratio test,LRT)结果同样支持上述推论。进一步利用经验贝叶斯法准确地检测出6 个受正选择作用的氨基酸位点(位点11、37、61、67、71、86),其中的5 个位点位于PBR 区。因此,正选择作用可能是维持黄麂DRB 基因多态性的主要机制之一。基于DRB 外显子2 序列利用邻接法(NJ)
构建了部分偶蹄动物系统发生关系,在NJ 树上,黄麂DRB 基因与其它鹿科动物DRB 基因呈镶嵌式分布,提示跨物种进化是维持黄麂DRB 基因多态性的另一重要机制。此外,黄麂两个等位基因(Mure-DRB1 和Mure-DRB11)和马鹿的两个等位基因(Ceel-DRB34 和Ceel-DRB46)与牛科的等位基因构成一个独立的进化枝,说明黄麂和马鹿的某些DRB 基因具有非常古老的谱系。     

Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP

In order to characterize the two new kappa-casein variants F and G (CSN3^F and CSN3^G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G→A) for CSN3^F and 10790 (C→T) for CSN3^G, corresponding to amino acid exchanges in positions 10 (Arg→His) and 97 (Arg→Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII , which were subsequently used to confirm these polymorphisms in cattle carrying CSN3^F or CSN3^G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3^A, CSN3^B, CSN3^C, CSNJ^E, CSN^F, CSN3^G) was developed.     

Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.     

Polymorphisms in MHC-DRA and -DRB alleles of water buffalo (Bubalus bubalis) reveal different features from cattle DR alleles

Seventy-five individuals of Bubalus bubalis belonging to four different breeds, three of river buffalo and one of swamp buffalo, were studied for polymorphism in MHC DRB (Bubu-DRB) and DRA (Bubu-DRA) loci. Eight alleles of Bubu-DRB were found, and all alleles in the swamp type were shared with the three river breeds. All alleles sampled from the breed of European origin (Mediterranean) were present in breeds sampled in Brazil, thus variability of this locus may have been preserved to a great extent in the more recently founded Brazilian population. Bubu-DRB alleles contained higher proportions of synonymous vs. non-synonymous substitutions in the non-peptide-binding sites (PBS) region, in contrast to the pattern of variation found in BoLA-DRB3, the orthologous locus in cattle. This indicated that either the first domain exon (exon 2) of Bubu-DRB has not undergone as much recombination and/or gene conversion as in cattle alleles, or Bubu-DRB may be more ancient than BoLA-DRB3 alleles. Phylogenetic analysis of DRB alleles from Bubalus, Syncerus c. caffer, the Cape buffalo, and domestic cattle demonstrated transspecies polymorphism. Water buffalo contained two alleles of DRA that differed from each other in two amino acid positions, including one in the PBS (alpha22) that was also shared with Anoa depressicornis, the anoa. Discovery of variation in DRA was surprising as the first domain of DRA is a highly conserved polypeptide in mammals in general and especially in ruminants, where no other substitution in PBS was seen.     

Multi-primer target PCR for rapid identification of bovine DRB3 alleles

Multi-primer target polymerase chain reaction (MPT-PCR) is a rapid method for the identification of specific BoLA-DRB3 alleles. In a single PCR reaction, the presence of two alleles associated with increased risk, DRB3.2*23 (DRB3*2701-2703, 2705-2707) and decreased risk, DRB3.2*16 (DRB3*1501, 1502), of mastitis in Canadian Holstein can be detected. Two outer primers amplify exon 2 of DRB3. Simultaneously, two inner, allele-specific primers amplify individual alleles. Initially, 40 cows previously typed by PCR-restriction fragment length polymorphism (PCR-RFLP) were genotyped using the multi-primer approach. An additional 30 cows were first genotyped by multi-primer target PCR, then by PCR-RFLP. All animals were correctly identified and there were no false positives. This technique can readily be modified to identify other BoLA alleles of interest.     

Polymorphism of the <Emphasis Type="Italic">BoLA-DRB3</Emphasis> gene in the Mongolian,Kalmyk, and Yakut cattle breeds

Polymorphism of the BoLA-DRB3 gene was studied with the use of the PCR-RFLP technique in three cattle breeds (Mongolian, Kalmyk, and Yakut) representing the Bos taurus turano-mongolicus group. 35 BoLA-DRB3.2 alleles were detected in the Mongolian breed and 34 alleles in the Kalmyk breed. The frequencies of alleles in both populations are distributed rather evenly: the frequencies of the most widely represented alleles (*18, *20, and *28) in the Mongolian cattle varied from 7.75 to 8.45%. The most frequent alleles in the Kalmyk cattle were *28 (14.52%), *24 (7.26%), and *12 (6.45%). Only five alleles were identified in the Yakut cattle breed. The prevailing allele was *29 (77.3%); a relatively frequent allele was *1 (13.1%), and the remaining three alleles constituted only 9.6%. Such a low level of diversity of BoLA-DRB3 gene alleles was not observed earlier in any other cattle breed. The Mongolian and Kalmyk breeds showed a wide diversity of BoLA-DRB3 genotypes (56 and 51 genotypes, respectively) and a high level of expected heterozygosity (H _e = 0.953 and 0.946, respectively). Both breeds had a deficiency of heterozygotes (Mongolian cattle: H _o = 0.775, D = −0.187; Kalmyk cattle: H _o = 0.708, D = −0.252). A low level of genotypic diversity for the BoLA-DRB3 locus (only seven genotypes; the frequency for the genotype *29/*29 is 71.4%) and a very low level of observed heterozygosity (H _o = 0.12) were revealed in the Yakut breed. BoLA-DRB3.2 alleles associated with resistance to persistent lymphocytosis caused by the bovine leukemia virus (total frequencies 15.49 and 24.19%) and to various forms of mastitis (total frequencies 12.68 and 20.96%, respectively) were identified in the Mongolian and Kalmyk animals. In the Yakut breed, alleles associated with resistance to diseases are represented only by the BoLA-DRB3.2 allele *7 (1.2%). Thus, the Mongolian and Kalmyk cattle breeds are characterized by a wide diversity of alleles and genotypes for the BoLA-DRB3 gene. In contrast, the population of Yakut cattle from the Verkhoyanskii region of the Republic of Sakha has a poor diversity of alleles and genotypes for the BoLA-DRB3 gene and a very low level of heterozygosity, suggesting an unfavorable state of the population that is probably caused by inbreeding depression due to a long-term isolation and a small number of animals.     

Association between BoLA-DRB3 and somatic cell count in Holstein cattle from Argentina

Different studies have proved that the resistance/susceptibility to mastitis is genetically determined. The major histocompatibility complex in cows is known as bovine lymphocyte antigen (BoLA). Genes from the BoLA have been associated with the occurrence of infectious diseases such as mastitis and leukosis, especially the BoLA-DRB gene. The object of the present study was to detect associations between BoLA-DRB3 alleles and somatic cell count (SCC), as an indicator of resistance/susceptibility to mastitis in Holstein cattle (N = 123) from La Pampa, Argentina. Fisher's exact test and Woolf-Haldane odds ratio were applied to study the association between SCC and BoLA-DRB3 allele frequencies. Significant association was noted between BoLA-DRB3.2*23 and *27 alleles (p < 0.05) and protective or susceptibility effects, respectively. In addition, alleles BoLA-DRB3.2*20 and *25 exhibit suggestive association with high SCC (p < 0.1). These results were partially in agreement with data reported from Japanese Holstein cattle, but differed from those published by other authors. A possible explanation for the contrasting results could be that the mastitis is a multifactor disease caused by different pathogens. Moreover, most of the studies were carried out using PCR-RFLP method, which has less resolution than PCR-SBT because PCR-RFLP defined alleles included more than one sequenced alleles.     

Screening of 12 SNPs of CYP3A4 in a Chinese population using oligonucleotide microarray

Human cytochrome P450 3A4 (CYP34A) plays an important role in the metabolism of many endo- and xenomaterials. It also exhibits a substantial interindividual variation in enzymatic activity. It has been shown that the mutant alleles of CYP3A4 encoding inactive/decreased enzymes are largely caused by single nucleotide polymorphisms (SNPs) in the gene sequence. In the present study, with the goal of detecting the known SNPs of CYP3A4, an oligonucleotide microarray was created. A genotyping standard for this microarray was also established using constructed plasmids as standard templates. The 12 SNPs of CYP3A4 in 387 Chinese DNA samples were screened using this oligonucleotide microarray. Three heterozygous subjects of CYP3A4*/*4, 5 heterozygous subjects of CYP3A4*1/*5, 4 heterozygous subjects of CPY3A4*1/6, and 6 heterozygous subjects of CYP3A4*1/*18 were found. The genotyping results of the 18 heterozygous subjects and 12 wild-type subjects were validated by direct sequencing.     

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.     

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.