Amplification and analysis of DNA sequences in single human sperm and diploid cells

Li, Honghua; Gyllensten, Ulf; Cui, Xiangfeng; Saiki, Randall K.; Erlich, Henry A.; Arnheim, Norman

doi:10.1038/335414a0

Cited by 620 publications

(233 citation statements)

References 12 publications

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“…Second, unlike yeast, where cells can be induced to synchronously enter sporulation and initiate meiosis (Govin and Berger 2009), meiotic progression is asynchronous in mammals and at any given moment <2% of the cells in the mouse gonads contain DSBs (Bellve et al 1977;Meistrich 1977). Unfortunately, sperm genotyping (Li et al 1988;Cui et al 1989;Hogstrand and Bohme 1994)-the method of choice for high-resolution recombination hotspot mapping in mammals-does not scale to genome-wide applications (for reviews, see Arnheim et al 2007;Kauppi et al 2009;Paigen and Petkov 2010).…”

mentioning

confidence: 99%

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Khil¹,

Smagulova²,

Brick³

et al. 2012

Genome Res.

114

139

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Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIPseq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method-single-stranded DNA (ssDNA) sequencing (SSDS)-that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

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mentioning

confidence: 99%

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Khil¹,

Smagulova²,

Brick³

et al. 2012

Genome Res.

114

139

View full text Add to dashboard Cite

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“…In vitro deoxyribonucleic acid (DNA) amplification by the polymerase chain reaction (PCR) makes it possible to detect rare DNA sequences [1], and can be used to identify the nucleic acids of infectious agents present in copy numbers that are too low to be detected by other methods [2]. This sensitive detection system has also been used for the identification of viral DNA in archival material, such as paraffin-embedded fixed tissue, where much of the DNA has been degraded and is no longer suitable for conventional methods of DNA analysis [3].…”

mentioning

confidence: 99%

Detection of adenovirus E1A DNA in pulmonary fibrosis using nested polymerase chain reaction

Kuwano¹,

Nomoto²,

Kunitake³

et al. 1997

Eur Respir J

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The history of patients with idiopathic pulmonary fibrosis (IPF)shows that the disease may be preceded by a viral-like illness. Although viruses have not been demonstrated, it is possible that viruses were not detected in culture because they do not replicate during latency.We investigated the presence of adenovirus in IPF and interstitial pneumonia associated with collagen vascular disease (CVD-IP), using the nested polymerase chain reaction (PCR) and in situ hybridization (ISH) for the E1A region of the adenovirus genome. Studies were performed on lung tissues obtained by transbronchial lung biopsy from 19 patients with IPF, 10 patients with CVD-IP and, for comparison, from 20 patients with sarcoidosis.The E1A DNA was present in 3 out of 19 (16%) cases of IPF, in 5 of 10 (50%) cases of CVD-IP, and in 2 of 20 (10%) cases of sarcoidosis. The incidence of E1A DNA in CVD-IP was significantly higher than that in sarcoidosis (p<0.05). In patients with IPF and CVD-IP, E1A DNA was more prevalent in patients treated with corticosteroids (6 out of 9 cases; 67%) than in those without it (2 out of 20 cases; 10%) (p<0.01). ISH studies showed that 1 out of 8 cases of IPF and CVD-IP, in which E1A DNA was detected by PCR, was positive for E1A DNA.We conclude that adenovirus E1A is unlikely to be aetiologically involved in the pathogenesis of idiopathic pulmonary fibrosis or interstitial pneumonia associated with collagen vascular disease. However, a latent adenovirus infection may be reactivated or may newly infect the host following corticosteroid administration.

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“…To inactivate proteinase K, the mixture was boiled for 5 min and then chilled on ice for 5 min. This mixture was used immediately in PCR (Li et al, 1988). …”

Section: Isolation and Characterization Of Genomic Clonesmentioning

confidence: 99%

Extreme heterogeneous composition of the Paramecium caudatum macronuclear genomic DNA between hemoglobin and nucleosome assembly protein-1 genes

et al. 2010

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The intergenic region between the hemoglobin (hb) and nucleosome assembly protein-1 (nap-1) genes in the Paramecium caudatum macronuclear genome was previously found to be heterogeneously composed. Cloning of this intergenic region from the macronuclear genomic DNA identified four unique DNA fragments of different sizes. Sequencing of the cloned fragments revealed extreme heterogeneity and characteristics of both internal eliminated sequence (IES) and imprecise internal deletion sequences (IIDSs) in the intergenic region. Missing sequences were an AT-rich and direct repeats existed in their boundaries. Southern blotting of the total genomic DNA and polymerase chain reaction (PCR) of the total genomic DNAs indicated that there exist a dozen DNA fragments of different sizes in this intergenic region. It is likely that the heterogeneity found in the P. caudatum macronuclear genome results from the variable removal of an intergenic region.

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Amplification and analysis of DNA sequences in single human sperm and diploid cells

Abstract: The use of the polymerase chain reaction for analysing DNA sequences in individual diploid cells and human sperm shows that two genetic loci can be co-amplified from a single sperm, which may allow the analysis of previously inaccessible genetic phenomena.

Cited by 620 publications

References 12 publications

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

Detection of adenovirus E1A DNA in pulmonary fibrosis using nested polymerase chain reaction

Extreme heterogeneous composition of the Paramecium caudatum macronuclear genomic DNA between hemoglobin and nucleosome assembly protein-1 genes

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