Multiplex PCR amplification of three microsatellites within the CFTR gene

Multiplex PCR amplification has been developed for three highly polymorphic microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) located in intronic regions of the CFTR (cystic fibrosis (CF) transmembrane conductance regulator) gene. The triplex PCR reaction required different concentrations of each pair of primers and labeling of primers in the same reaction. Total informativity is obtained in 90.25% of couples requiring analysis of polymorphisms, and when triplex microsatellite analysis is combined with analysis for the six most common CF mutations in the Spanish population, informativity reaches more than 99%.     

Linkage disequilibrium and founder effect analysis of the NF1 gene in French Canadians from the Quebec population

We genotyped 19 neurofibromatosis type 1 (NF1) families from French Canadians of the Quebec population with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Linkage analysis of the four microsatellite markers among the 19 NF1 families indicates that the four microsatellites are strongly linked with NF1 disease (LOD = 2.76-3.64). The four markers are associated (P = 0-0.077) except marker pair IVS26-2.3/IVS27AC33.1 (P = 0.18 or 0.17). However, perhaps due to the high mutation rate of the NF1 gene, no founder effect for NF1 was detected in the Quebec French Canadians.     

Amplifying dinucleotide microsatellite loci from bone and tooth samples of up to 5000 years of age: more inconsistency than usefulness

We have studied the feasibility of using dinucleotide-repeat microsatellites in the analysis of DNA from ancient bones and teeth. We have used three microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) within the cystic fibrosis transmembrane conductance regulator gene in 28 DNA samples from bones and teeth of up to 5000 years of age. PCR amplification was successful in 71.4% of cases. The repeated analysis of each marker produced different genotypes in 97% of samples, and the same individual genotype was reproduced at least once in 45.5% of cases. Alleles differing from the originals consisted of additions or deletions of 1–39 dinucleotides. The mechanism by which alleles differing from the originals were amplified can be related to the marked degradation of the DNA, with repeat sequences of different length interacting with the partially degraded repeats of the amplified loci. The repeated analysis of each sample allowed us to produce data with some anthropological interest. Among the haplotypes detected in samples from Easter Island, two (16-32-13 and 23-32-13) were found in more than one sample. Similarly, three haplotypes (16-7-17, 16-7-13, and 16-24-13) were detected more than once in samples from the Basque Country. Although haplotypes in the Basque Country are amongst the commonest in European chromosomes, most of those detected in the Easter Island samples are not frequent in Europeans. Thus, the repeated typing of microsatellites allowed us to postulate the genotypes that might be present in the samples but dinucleotide markers do not seem to be reliable enough for genotyping ancient bone and teeth samples.     

Analysis of Two Polymorphic Repeats IVS3 Poly A and IVS10 CA in Tunisian Cystic Fibrosis Patients: Case–Control Study

Russian Journal of Genetics - The aim of this study was to determine a possible association of IVS3 poly A and IVS10 CA microsatellites with CF in case–control Tunisian groups and to compare...     

Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism

In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA. Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing exons 13 – 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two short direct repeats flanking the breakpoints. The 3′ repeat partially overlapped the IVS17b/CA microsatellite and the number of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed. These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during DNA replication, which has not previously been described in the CFTR gene. Received: 27 December 1995 / Accepted: 30 January 1996     

Analysis of intragenic polymorphic markers of the CFTR gene in cystic fibrosis patients and health donors from Bashkorostan

The differences in the polymorphic allele frequency distribution patterns of the biallelic (M470 and TUB20) and microsatellite (IVS6aGATT, IVS8CA, and IVS17CA) markers within the CFTR gene between normal and delF508 chromosomes have been established. For most of the marker loci similar distribution of the allele frequencies on normal and mutant chromosomes without delF508 was demonstrated. Certain polymorphic alleles displayed substantial linkage disequilibrium with the delF508 mutation. Analysis of the IVS6aGATT-IVS8CA-M470-IVS17CA-TUB20 haplotypes association on normal and mutant chromosomes provided identification of the delF508 ancestral haplotype. It was suggested that delF508 mutant chromosomes were introduced into the modern Bashkir gene pool as a result of Slavic migrations from the Eastern Europe. The IVS6aGATT-IVS8CA-M470-IVS17CA-TUB20 major haplotype (77272) revealed was statistically significantly most frequently found on the mutant chromosomes without the delF508 mutation. This finding suggests that the Bashkir cystic fibrosis patients, mostly belonging to the Turkic-speaking families, possessed specific CF gene defect associated with the given haplotype.     

Cystic fibrosis in Spain: high frequency of mutation G542X in the Mediterranean coastal area

We have determined the frequency of deletion F508 and mutation G542X, a nonsense mutation in exon 11 of the cystic fibrosis (CF) gene, in a sample of 400 Spanish CF families. Mutation G542X represents 8% of the total number of CF mutations in Spain, making it the second most common mutation after the F508 deletion, which accounts for 48% of CF chromosomes. G542X has a higher frequency in the Mediterranean coastal area (14%) and in the Canary Islands (25%). About 70% of G542X chromosomes are from Andalucia, Múrcia, Valencia, Catalunya and the Canary Islands. The F508 deletion has its highest frequency in the Basque Country (83%). Mutation G542X is associated with the same rare haplotype that is found in association with the F508 mutation. The haplotype homogeneity found for G542X, even when intragenic microsatellites (IVS8CA, IVS17BTA and IVS17BCA) are considered, allows us to postulate that this mutation arose from a single mutational event. The geographic distribution of mutations F508 and G542X suggests that F508 was present in the Iberian Peninsula before the Indo-European invasions, and that G542X was introduced into Spain, via the Mediterranean Sea, probably by the Phoenicians, between 2500 and 3000 years ago.     

Isolation and characterization of 17 polymorphic microsatellites in grass carp

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.     

Analysis of the Polymorphic Markers within the CFTR Gene in Cystic Fibrosis Patients and Healthy Donors from Bashkortostan

The differences in the polymorphic allele frequency distribution patterns of the biallelic (M470V and TUB20) and microsatellite (IVS6aGATT, IVS8CA, and IVS17CA) markers within the CFTR gene between normal and delF508 chromosomes have been established. For most of the marker loci similar distribution of the allele frequencies on normal and mutant chromosomes without delF508 was demonstrated. Certain polymorphic alleles displayed substantial linkage disequilibrium with the delF508 mutation. Analysis of the IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20 haplotypes association on normal and mutant chromosomes provided identification of the delF508 ancestral haplotype. It was suggested that delF508 mutant chromosomes were introduced into the modern Bashkir gene pool as a result of Slavic migrations from the Eastern Europe. The IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20major haplotype (77272) revealed was statistically significantly most frequently found on the mutant chromosomes without the delF508 mutation. This finding suggests that the Bashkir cystic fibrosis patients, mostly belonging to the Turkic-speaking families, possessed specific CFTR gene defect associated with the given haplotype.     

CFTR haplotypic variability for normal and mutant genes in cystic fibrosis families from southern France

In order to contribute to a better understanding of the dispersion of cystic fibrosis (CF) mutations in the South of France, seven diallelic and three multiallelic markers [three upstream of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (XV-2c, KM.19 and J44) and seven intragenic polymorphisms (IVS6A, IVS8CA, M470V, T854T, IVS17BTA, IVS17BCA and TUB18)] were analyzed for 143 ΔF508 chromosomes, 100 CF chromosomes carrying 85 non-ΔF508 and 15 unknown mutations, and 198 normal CFTR alleles. The study provides haplotypic data for 39 different CF mutations, which should be useful in diagnosis by haplotypic analysis and detection of the associated mutations. A major haplotype [2-1-2-7-16-2-1-(30/31)-13-1] was found in normal chromosomes, which should be the most ancient in the Caucasoid population. The most frequent haplotypes in normal chromosomes were associated with 16 different non-ΔF508 mutations, suggesting that there was no preferential haplotype on which these mutations arose. Several mutations were each associated with more than one haplotype, as the result of slippage at one or two of the three microsatellites (ΔF508, G542X, N1303K, G85E, E585X, K710X and 2184delA) or recombination (1717-1G→A, R334W, L206W, R1162X and Y122X). Haplotypes for the most common CFTR mutations (ΔF508, G542X, N1303K) revealed that a large number of alleles were generated by slippage at the microsatellite loci, suggesting that they are the most ancient CF mutations. Other mutations were associated with haplotypes that were different either at several diallelic sites (R334W) or at both diallelic and microsatellite markers (R1162X and R1158X), which is more suggestive of recurrence. Twenty recombinations were detected among the CF mutant alleles analyzed, 75% of them occurring in the second half of the CFTR gene. The higher mutational heterogeneity and the haplotypic variability reported in this small population from the Mediterranean area are consistent with an earlier appearance of CFTR mutations in southern Europe than in central and northern Europe, and an earlier origin and expansion of this population. Received: 19 February 1996 / Revised: 10 April 1996     

GenBank数据库中鳜鱼微卫星位点的筛选及特征分析

微卫星(Microsatellite)是一类由2-6个核苷酸经多次单位串联组成的高度变异重复DNA序列(Schlotterer and Tautz,1992)。它具有按照孟德尔方式分离、突变快、多态信息含量丰富、呈共显性遗传等特点,其核心序列在同一物种中具有保守性,因此,可以根据微卫星的侧翼序列设计合适的引     

SARS冠状病毒S蛋白候选细胞受体氨基肽酶N的基因变异研究

氨基肽酶N是人类冠状病毒HCoV 2 2 9E的细胞受体 ,可能为SARS冠状病毒 (SARS CoV)S蛋白的受体。应用变性高效液相色谱 (DHPLC)技术筛查了正常无关个体中氨基肽酶N编码基因ANPEP的全部外显子及其两侧部分内含子序列。对筛查中DHPLC峰型提示有基因变异存在的DNA片段行PCR产物直接测序 ,共发现 9种单核苷酸多态 (SNP) ,其中 4种为非同义SNP ,分别为T32 1M(96 2C >T)、S6 5 1L(195 2C >T)、S75 2N(2 2 5 5G >A)和G76 4R(2 2 90G >A) ;其余 5种SNP为T795T(2 385C >T)、IVS7+17G >A、IVS14 16A >G、IVS17+12C >G和IVS17+44C >T。这些SNP的发现 ,为SARS CoV的宿主遗传因素研究 ,特别是发现SARS CoV感染和SARS发病的易感基因或抗病基因 ,提供了遗传标记。     

Chloroplast microsatellites in Prunus,Rosaceae

Ten chloroplast microsatellite markers were developed from Japanese plum (Prunus salicina) based on nucleotide sequences of c. 4300 bp from six chloroplast regions. Out of 10 microsatellites, seven markers contained mononucleotide repeats. Almost all microsatellites displayed discrete amplified fragments for 17 species in Prunus. The microsatellites generated 46 different fragment types and differentiated all used species. Polymorphism was also observed within species for all microsatellites.     

Characterization of EST-derived microsatellites for gene mapping and evolutionary genomics in turbot

The detection of microsatellite sequences within expressed sequence tags (ESTs) connects potential markers with specific genes, generating type I markers. We have developed and mapped by linkage analysis a set of EST-derived microsatellites in the turbot, Scophthalmus maximus. One hundred and ninety-one microsatellites were identified from 9256 turbot ESTs. Primer design was possible with 98 microsatellites. After genotyping 25 wild turbot and the parents of two reference families for linkage analysis, 43 EST-derived microsatellites were selected because they met technical and polymorphism criteria. A final set of 31 EST-derived microsatellites could be mapped to 17 linkage groups of the turbot consensus map based on 242 anonymous microsatellites. Twenty-four microsatellite-containing ESTs were functionally annotated, confirming them as type I markers. Nineteen were mapped in the turbot consensus map. These EST-derived microsatellites constitute useful tools for genome scanning of turbot populations, marker-assisted selection programmes and comparative mapping.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Dinucleotide (CA/GT) repeat polymorphism in intron 17B of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Summary We describe a polymorphic microsatellite (IVS17BCA) in intron 17B of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It consists of an 11 to 20 CA/GT dinucleotide repeat, located 424 bp from exon 17B.     

Evaluation of ACE, SP17, and FSHB as candidates for stallion fertility in Hanoverian warmblood horses

The research of fertility in humans and other mammals has strongly advanced in the recent years. The examination of molecular mechanisms influencing horse fertility is relatively recent. We chose the angiotensin converting enzyme (ACE), the sperm autoantigenic protein 17 (SP17) and the follicle stimulating hormone (FSHB) as candidates for determining stallion fertility and to analyze associations of intragenic single nucleotide polymorphisms (SNPs), flanking microsatellites and candidate-gene linked haplotypes with the pregnancy rate per oestrus (PRO) in 179 Hanoverian stallions. Fertility traits analyzed were the least square means of PRO for stallions (LSMs) and the paternal and embryonic component of breeding values for PRO (BVs). We detected nine SNPs and two flanking microsatellites in ACE, eight SNPs and two flanking microsatellites in SP17 and four SNPs and one flanking microsatellite in FSHB. Three SP17-associated SNPs and the two flanking microsatellites showed significant association with the embryonic component of BVs and one SP17-associated microsatellite was also significantly associated with the paternal component of BVs. Two ACE-associated SNPs were significantly associated with the embryonic component of BVs. Significantly associated haplotypes were shown for all three candidate genes and the tested fertility parameters. The final regression analysis model indicated that haplotypes of all three candidate genes significantly contributed to the paternal and embryonic fertility components of PRO. This is the first report of associations of ACE, SP17 and FSHB with fertility traits of stallions.     

Low abundance of microsatellite repeats in the genome of the brown-headed cowbird (Molothrus ater).

A cosmid library made from brown-headed cowbird (Molothrus ater) DNA was examined for representation of 17 distinct microsatellite motifs including all possible mono-, di-, and trinucleotide microsatellites, and the tetranucleotide repeat (GATA)n. The overall density of microsatellites within cowbird DNA was found to be one repeat per 89 kb and the frequency of the most abundant motif, (AGC)n, was once every 382 kb. The abundance of microsatellites within the cowbird genome is estimated to be reduced approximately 15-fold compared to humans. The reduced frequency of microsatellites seen in this study is consistent with previous observations indicating reduced numbers of microsatellites and other interspersed repeats in avian DNA. In addition to providing new information concerning the abundance of microsatellites within an avian genome, these results provide useful insights for selecting cloning strategies that might be used in the development of locus-specific microsatellite markers for avian studies.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.