Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Findlay, lan; Ray, Pierre F.; Quirke, Phil; Rutherford, Anthony J.; Lilford, Richard

doi:10.1093/molehr/1.4.209

Cited by 67 publications

(97 citation statements)

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“…For instance, this effect has been detected in DNA amplifications from both single cells and human blastomeres [26,27] and it was recently reported in the amplification of the CEB205 minisatellite locus [28]. Here, we have shown that PCR buffers containing 50 mM KC1 can lead to preferential amplification of the allele 73.1, but not of 80.1.…”

Section: Discussionsupporting

confidence: 64%

Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

et al. 2004

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PCR preferential amplification consists of the inefficient amplification of one allele in a heterozygous sample. Here, we report the isolation of a GC-rich human minisatellite, MsH43, that undergoes allelic preferential amplification during PCR. This effect requires the existence of a (TGGGGC) 4 motif that is able to form a G-quadruplex in the presence of K þ . This structure interferes with the DNA synthesis of the alleles harbouring this motif during PCR The present results are the first demonstration that the formation of G-quadruplex can be one of the mechanisms involved in some kinds of preferential amplification.

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Section: Discussionsupporting

confidence: 64%

Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

et al. 2004

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“…Unique clones originate from different sperm cells and may be more representative of the methylation status of different sperm cells and the sample. Preferential amplification may occur when small quantities of starting material are used (Walsh et al 1992, Findlay et al 1995; therefore to prevent this from occurring, multiple amplification reactions were set up per gene for each sample and unique clones were analyzed.…”

Section: Analysis Of Sequencing Datamentioning

confidence: 99%

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Minor¹,

Chow²,

Ma³

2011

Reproduction

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Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (NZ18), including OA (NZ10), NOA (NZ5), and unknown pathology (NZ3), and from men undergoing vasectomy reversal (NZ17). Sperm was also obtained from proven fertile men (NZ9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, IG-GTL2 and MEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation at IG-GTL2 and MEST DMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at the H19 DMR was particularly prone to modification in testicular sperm.

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“…ADO can be defined as amplification failure affecting only one of the parental alleles present in the single cell [10]. ADO's incidence varies, but in extreme cases has affected 20% of amplifications [11][12][13][14], and in the past has led to several misdiagnoses [15][16][17].…”

Section: Diagnosis Of Single Gene Defectsmentioning

confidence: 99%

Preimplantation genetic diagnosis: present and future

Fragouli

2007

J Assist Reprod Genet

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Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Cited by 67 publications

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Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Preimplantation genetic diagnosis: present and future

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Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Cited by 67 publications

References 0 publications

Inhibition of DNA synthesis by K+‐stabilised G‐quadruplex promotes allelic preferential amplification

Inhibition of DNA synthesis by K+‐stabilised G‐quadruplex promotes allelic preferential amplification

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Preimplantation genetic diagnosis: present and future

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Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification