Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

Li, Honghua; Cui, Xiangfeng; Arnheim, Norman

doi:10.1073/pnas.87.12.4580

Cited by 117 publications

(54 citation statements)

References 16 publications

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“…Since efficiency of sexing decreased significantly when less than five cells were subjected to PCR [52], the larger sampling size (8-10 cells) has been recommended for successful sexing of the embryos [53]. However, development of "primer extension preamplification-PCR (PEP-PCR)", which is an in vitro-system for amplifying a large fraction of the DNA sequence present in a single haploid cell [54,55], made it possible to identify the sex of embryos using a single blastomere. The application of PEP-PCR to bovine embryo sexing resulted in a 91% sexidentification rate [56].…”

Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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“…These include M. cryptica and M. nubilosa, the two most common and destructive pathogens on E. globulus in southern Australia, which were distinguished both in culture and as in infected leaf tissue. Detection of the presence of a species in a leaf, however, does not mean that it is causing a high level of infection, as PCR is extremely sensitive, being able to detect even a single molecule of template DNA (Li, Cui & Arnheim 1990). amplified rDNA sequences from a single spore of Neurospora tetrasperma.…”

Section: Discussionmentioning

confidence: 99%

A specific primer PCR and RFLP assay for the rapid detection and differentiation in planta of some Mycosphaerella species associated with foliar diseases of Eucalyptus globulus

Kularatne

Lawrie

Barber

et al. 2004

Mycological Research

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It is difficult to accurately identify Mycosphaerella species associated with leaf diseases of Eucalyptus based on morphological characters, as there is considerable overlap between very similar species and subspecies, and isolation from the host is not easy. Thus, a PCR and RFLP assay based on the ITS region of nr DNA was developed for the rapid detection and differentiation of M. nubilosa, M. cryptica and two non-sporing unidentified Mycosphaerella species isolated from the foliage of trees in resistant and susceptible families of E. globulus in a seed orchard at Kinglake West, Victoria, Australia. The M. nubilosa primer pair MNF/MNR was highly specific. A PCR-RFLP system based on the primer pair MCF/MCR, coupled with two restriction enzymes (DdeI and Tru1I), differentiated M. cryptica, M. nubilosa, M. tasmaniensis and M. aff. vespa. One of the unidentified field-isolated Mycosphaerella species was identified as M. grandis on the basis of ITS sequence data while the other species remains unidentified. A PCR-RFLP system based on the primer pair U1F/U1R, coupled with the restriction enzyme StyI, differentiated between the two unidentified species. Unexpectedly, unlike isolation and culture studies, these assays detected M. nubilosa, M. cryptica and M. grandis in all single lesions examined on both juvenile and adult leaves, and on both highly resistant and highly susceptible E. globulus trees at this site.

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“…22 Compared with the single-round PCR protocol in which two primers are used, our two-round PCR protocol involves using three primers. Because the presence of genomic DNA and formation of "primer-dimers," single-round PCR is often associated with a significant amount of non-specific amplification.…”

Section: Using Nested Primers With a Two-round Amplification Protocolmentioning

confidence: 99%

Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

Cui

Feiner

2002

The Journal of Molecular Diagnostics

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Loss of heterozygosity (LOH) is an indication of tumor suppressor gene inactivation. However, loss of heterozygosity analysis has been limited to either a small scale or to very few genetic markers. To significantly increase the scale of study and to include a large number of markers in the analysis, experimental conditions were established for using single cells or single cell equivalent with 10 markers typed simultaneously. Under these conditions, the allele amplification failure rate was 3.7% when single tissue cultured human cells were used. When 30 cells from a 5-m paraffin-archived breast tumor tissue section were used, the failure rates were 0% for four of the five heterozygous loci and 10% for the fifth. Small amplification failure rates (6.1% and 6.7% on average) were observed when 5 or 10 cells from paraffin-archived breast tissue were used. These results indicate that with polymerase chain reaction (PCR) primers of high quality, it is possible to obtain reliable results by using single cells from fresh tissue or very few cells from paraffin-archived specimens. The results also show the importance of including replicates, using primers of high quality, and optimizing PCR conditions when a limited amount of material is used for the assay.

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Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

Cited by 117 publications

References 16 publications

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

A specific primer PCR and RFLP assay for the rapid detection and differentiation in planta of some Mycosphaerella species associated with foliar diseases of Eucalyptus globulus

Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

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