scholarly journals Human mutations and their detection by gene and linkage analysis, allele sharing and association methods

1999 ◽  
Vol 5 (6) ◽  
pp. 1140-1146
Author(s):  
J. A. Phillips ◽  
R. Hamid
Keyword(s):  
Linkage Analysis ◽  
Dna Analysis ◽  
Direct Detection ◽  
Polymerase Chain Reaction Amplification ◽  
Point Mutations ◽  
Indirect Detection ◽  
Dna Polymorphisms ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Detection Of Mutations

Ithas been 20 years since DNA analysis was first used in the detection of sickle-cell anaemia. Here, techniques for detecting human mutations are reviewed. We describe direct detection of mutations using restriction enzyme analysis and polymerase chain reaction amplification to detect gene deletions, rearrangements and point mutations. Indirect detection of mutations include the use of DNA polymorphisms in linkage analysis

Characterization and rapid diagnostic analysis of DNA polymorphisms closely linked to the cystic fibrosis locus

1990 ◽  
Vol 36 (9) ◽  
pp. 1614-1619 ◽  
Author(s):  
G T Horn ◽  
B Richards ◽  
J J Merrill ◽  
K W Klinger
Keyword(s):  
Cystic Fibrosis ◽  
Linkage Analysis ◽  
Direct Detection ◽  
Pcr Amplification ◽  
Dna Polymorphisms ◽  
Cystic Fibrosis Gene ◽  
Alu Repeat ◽  
Allele Specific ◽  
Polymorphic Restriction ◽  
Detection Of Mutations

Abstract Six genetic polymorphisms, closely linked to the cystic fibrosis gene and useful in clinical linkage analysis, have been characterized and converted to a more rapid form of assay. Sequences flanking the metD (Ban I), metH (Msp I), XV-2c (Taq I), KM.19 (Pst I), MP6d-9 (Msp I), and J3.11 (Msp I) polymorphic restriction sites have been determined and used to design specific polymerase chain reaction (PCR) amplification primers and allele-specific oligonucleotide probes. All six of these polymorphisms were found to involve single-base alterations, and the XV-2c polymorphism was found to lie within an Alu repeat segment. These PCR-based tests, in conjunction with the CS.7 (Hha I) assay described elsewhere (Stanier P et al. Hum Genet 1988;80:309-10; Williams C et al. Lancet 1988;ii:102-3), provide a convenient, rapid, and reliable method of haplotype and linkage analysis, clinically useful in those situations where direct detection of mutations is not possible.

scholarly journals Oligonucleotide microarray based method for simultaneous diagnostic of 3-M syndrome, SOPH syndrome, tyrosinemia type 1, methaemoglobinaemia type 1, nonsyndromic hearing loss and deafness (DFNB1)

2019 ◽  
pp. 24-33
Author(s):  
М.Т. Саввина ◽  
А.Л. Сухомямова ◽  
П.И. Голикова ◽  
А.Л. Данилова ◽  
Н.Р. Максимова
Keyword(s):  
Hearing Loss ◽  
Dna Microarray ◽  
Hereditary Diseases ◽  
Genetic Diagnostics ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Nonsyndromic Hearing Loss ◽  
Tyrosinemia Type 1 ◽  
Detection Of Mutations

Актуальность. В популяции якутов Республики Саха (Якутия) по данным ряда авторов наблюдается значительное накопление наследственных заболеваний. Частота некоторых заболеваний выше, чем в России и мире. В республике стоит необходимость проведения мероприятий по снижению генетического груза путём проведения скрининга населения. Существующие в настоящее время методы генетической диагностики имеют ограничения из-за ресурсозатратности и продолжительного времени проведения одного анализа. Одним из перспективных методов ДНК-диагностики, позволяющим одновременно проводить определение нескольких точковых мутаций, является анализ на биочипах, имеющий высокую скорость проведения исследования и высокую достоверность получаемых результатов. Цель работы - разработка способа одновременной детекции носительства мутаций частых наследственных заболеваний у населения якутской этнической группы с использованием ДНК-биочипа. Методы. Для формирования диагностической панели биочипа были проанализированы научная литература и генетический регистр наследственных болезней МГЦ НЦМ РБ №1. Синтетические олигонуклеотиды наносились на стеклянную подложку биочипа бесконтактным способом печатающей станцией Perkin Elmer Piezzorray (Perkin Elmer, США). Диагностика на биочипе основана на реакции обратной гибридизации и включает два этапа мультиплексной ПЦР с наработкой флуоресцентно-меченых одноцепочных продуктов с последующей их гибридизацией с мишенями на подложке. Верификация результатов генотипирования на биочипе проводилась методами ПЦР-ПДРФ и ПЦР в реальном времени. Результаты. Нами разработан ДНК-биочип для выявления мутаций в генах CUL7, NBAS, FAH, DIA1 и GJB2, которые являются причиной возникновения пяти наследственных болезней: 3-М синдрома, SOPH-синдрома, тирозинемии 1 типа, наследственной энзимопенической метгемоглобинемии I типа, наследственной несиндромальной глухоты 1А типа соответственно. Были проведены испытания экспериментальных образцов биочипов, а также сравнение полученных данных с результатами ДНК-диагностики исследуемых наследственных заболеваний на той же выборке образцов классическими методами. На данный способ диагностики получен Патент РФ «Способ одновременной диагностики наследственных заболеваний» №2627115 от 03.08.2017 г. Background and objectives. High incidence and high prevalence of hereditary diseases in Yakut ethnic group of Republic of Sakha (Yakutia) has been reported by several research groups. The incidence of some of the diseases are much higher comparing to data in other regions of Russia and the worldwide. There is a pressing need for prevention of those diseases through the mass genetic screening of population. The methods of genetic diagnostics that are known today are not able to afford the mass screening due to the high cost and time consumption for a single test. One of promising technologies and methods of DNA diagnostics is DNA microarray. It enables researchers to quickly screen large numbers of biological analytes for a variety of purposes including disease diagnostics. Methods. A Perkin Elmer Piezzorray (Perkin Elmer) microarrayer was used to print oligonucleotide based low-density microarrays by non-contact manner for addressing 5 mutations causing 5 frequently occurring diseases reported from yakut population. The assay using developed microarray chip based on reverse hybridization which include two step multiplex PCR reactions with production of one-stranded Cy5 labeled-PCR products following its hybridization with oligonucleotide probes. Results. In this study we developed a DNA microarray for detection of mutations in genes CUL7, NBAS, FAH, DIA1, GJB2 genes which are known to be cause of five very frequently occurring hereditary diseases in republic of Sakha (Yakutia): 3-M syndrome, SOPH-syndrome, tyrosinemia type 1, methaemoglobinaemia type 1, nonsyndromic hearing loss and deafness (DFNB1) type 1A respectively. Testing of experimental versions of DNA chips as well as control DNA diagnostic of five hereditary diseases on the same DNA samples using PCR with PCR and restriction enzyme analysis and real-time PCR were carried out.

scholarly journals Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines

2004 ◽  
Vol 70 (3) ◽  
pp. 1347-1355 ◽  
Author(s):  
Luca Cocolin ◽  
Kalliopi Rantsiou ◽  
Lucilla Iacumin ◽  
Roberto Zironi ◽  
Giuseppe Comi
Keyword(s):  
Direct Detection ◽  
Molecular Methods ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Brettanomyces Bruxellensis ◽  
Dna And Rna ◽  
Detection And Identification ◽  
26S Rrna ◽  
Total Agreement

ABSTRACT In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

scholarly journals A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

2021 ◽  
Vol 62 (4) ◽  
pp. 371-377
Author(s):  
Rossana C. Jaspe ◽  
Yoneira Sulbaran ◽  
Mariana Hidalgo ◽  
Mariana Hidalgo ◽  
Carmen L. Loureiro ◽  
...  
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Restriction Enzyme ◽  
Restriction Analysis ◽  
Spike Protein ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Simple Method ◽  
Spike Gene ◽  
Detection Of Mutations

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

scholarly journals Molecular characterization of antigenic polymorphisms (Ond(a) and Mart(a)) of the beta 2 family recognized by human leukocyte alloantisera

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
S Simsek ◽  
CE van der Schoot ◽  
M Daams ◽  
E Huiskes ◽  
M Clay ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutations ◽  
Human Leukocyte ◽  
Enzyme Analysis ◽  
Mononuclear Leukocytes ◽  
Restriction Enzyme Analysis ◽  
Coding Region ◽  
Amino Acid Polymorphism ◽  
Dna Coding ◽  
Beta 2

We show that the previously described alloantisera Ond and Mart, which recognize the alloantigens Ond(a) and Mart(a), react with polymorphic variants of alpha L and alpha M subunits of the beta 2 integrin family (CD11a and CD11b molecules). This was shown by testing the alloantisera in a monoclonal antibody-specific immobilization of leukocyte antigens, immunoprecipitation, and immunofluorescence assay against cells from normal donors and from patients with leukocyte adhesion deficiency (beta 2 intergrin deficient). To elucidate the molecular basis of the Ond(a) and Mart(a) alloantigens, RNA was isolated from mononuclear leukocytes derived from individuals of known serologic phenotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify the entire coding region of the alpha L and alpha M mRNAs. The Ond(a) antigen was found to be due to a G2466C substitution in the DNA coding for the alpha L subunit, which predicts an Arg766Thr amino- acid polymorphism. The Mart(a) antigen was also found to be due to a single nucleotide substitution (G302A) in the DNA coding for the alpha M subunit, which predicts an Arg61His amino acid polymorphism. Using allele-specific restriction enzyme analysis, the association between point mutations and phenotypes was confirmed. The localization of these alloantigens on integrin molecules further illustrates the polymorphic nature of this class of proteins. Whether the polymorphisms influence the adhesive capacity of the leukocyte integrins remains to be investigated.

Carrier detection and prenatal diagnosis in 98 families of haemophilia A by linkage analysis and direct detection of mutations

1991 ◽  
Vol 2 (2) ◽  
pp. 293-302 ◽  
Author(s):  
J. M. Lavergne ◽  
Y. Laurian ◽  
A. Dudilleux ◽  
M. J. Larrieu ◽  
B. R. Bahnak ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Linkage Analysis ◽  
Direct Detection ◽  
Carrier Detection ◽  
Haemophilia A ◽  
Detection Of Mutations

scholarly journals Importance of mutations in amino acid 484 of the Spike protein of SARS-CoV-2: rapid detection by restriction enzyme analysis

2021 ◽  
Vol 62 ◽  
pp. 18-26
Author(s):  
Rossana C Jaspe ◽  
Yoneira Sulbarn ◽  
Carmen L Loureiro ◽  
Pierina D´Angelo ◽  
Lieska Rodríguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Enzyme ◽  
Rapid Detection ◽  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Spike Gene ◽  
Detection Of Mutations ◽  
Viral Isolates

Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 484 of the Spike protein are particularly important and associated with some of these variants: E484K or E484Q. These mutations have been associated with evasion to neutralizing antibodies. Restriction enzyme analysis is proposed as a rapid method to detect these mutations. A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. E484K, present in some VOCs, has emerged in several other lineages and is frequently found in recent viral isolates. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, and MseI. The use of these two enzymes allows the detection of mutations at position 484, and to differentiate between these three conditions: non-mutated, and the presence of E484K or E484Q. A 100% correlation was observed with sequencing results. The proposed methodology, which allows for the screening of a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.

Variation in natural populations of Drosophila as revealed by four-cutter analysis

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 784-787 ◽  
Author(s):  
Montserrat Aguadé
Keyword(s):  
Restriction Site ◽  
Natural Populations ◽  
Restriction Enzymes ◽  
Dna Polymorphisms ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Molecular Variation ◽  
Deletion Polymorphism ◽  
Isolated Populations ◽  
Chromosome Arrangements

Four-cutter restriction enzyme analysis, a recently developed electroblotting technique, enables the survey of restriction site and insertion–deletion polymorphism in natural populations at a fine level of resolution. Using this technique, the distribution of polymorphism among geographically isolated populations of Drosophila melanogaster and in different structural–functional domains of the genome has been studied. A summary of these results is presented and discussed. Recent investigations of molecular variation within and between different chromosome arrangements in Drosophila are presented. Levels of variation in different regions of the X chromosome of D. melanogaster are also discussed.Key words: populations, DNA polymorphisms, Drosophila, restriction enzymes.

scholarly journals Comparative Analysis and Serovar-Specific Identification of Multiple-Banded Antigen Genes of Ureaplasma urealyticum Biovar 1

1999 ◽  
Vol 37 (3) ◽  
pp. 538-543 ◽  
Author(s):  
Fanrong Kong ◽  
Xuejun Zhu ◽  
Weizhen Wang ◽  
Xiangzhao Zhou ◽  
Susanna Gordon ◽  
...  
Keyword(s):  
Sex Workers ◽  
Gene Sequence ◽  
Ureaplasma Urealyticum ◽  
Direct Detection ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Specific Identification ◽  
Clinical Specimens ◽  
Detection And Identification ◽  
Nongonococcal Urethritis

Ureaplasma urealyticum is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant U. urealyticum antigen recognized during infections in humans and probably has a significant role in virulence. The 5′ end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5′ ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5′ end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125–UMA269 and UMS-125–UMA269′. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing U. urealyticumbiovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic (P < 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing U. urealyticum biovar 1.

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