scholarly journals Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination

2001 ◽  
Vol 7 (5) ◽  
pp. 489-494 ◽  
Author(s):  
P. F. Ray ◽  
M. Vekemans ◽  
A. Munnich
Keyword(s):  
Multiplex Pcr ◽  
Single Cell ◽  
Pcr Amplification ◽  
Dystrophin Gene ◽  
Gender Determination

scholarly journals New Male Specific Markers for Hop and Application in Breeding Program

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andreja Čerenak ◽  
Zala Kolenc ◽  
Petra Sehur ◽  
Simon P. Whittock ◽  
Anthony Koutoulis ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Low Cost ◽  
Pcr Amplification ◽  
Breeding Program ◽  
Phenotypic Data ◽  
World Collection ◽  
Crude Sample ◽  
Male Sex ◽  
Male Specific

Abstract Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94–100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR

2007 ◽  
Vol 19 (1) ◽  
pp. 300
Author(s):  
M. Zhang ◽  
Q. Fu ◽  
W. S. Qin ◽  
H. Y. Zheng ◽  
Y. Q. Lu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Pcr Amplification ◽  
Single Copy ◽  
Bubalus Bubalis ◽  
Sex Identification ◽  
Sry Gene ◽  
Embryo Sex ◽  
Pcr Method ◽  
Male Specific

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos. This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).

scholarly journals Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell

2003 ◽  
Vol 49 (3) ◽  
pp. 415-424 ◽  
Author(s):  
James M Gale ◽  
Christopher P Romero ◽  
Gregory B Tafoya ◽  
Jérôme Conia
Keyword(s):  
Dna Sequencing ◽  
Single Cell ◽  
Optical Trapping ◽  
Pcr Amplification ◽  
P53 Gene ◽  
Rrna Gene ◽  
Dna And Rna ◽  
A Cell ◽  
Nonadherent Cells ◽  
Positive Results

Abstract Background: Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR. Methods: Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing. Results: Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)n length polymorphism in p53 introns. Conclusions: These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.

scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

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