Dataset Information

An Assay to Study Intra-Chromosomal Deletions in Yeast.

ABSTRACT: An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem direct repeats in Schizosaccharomyces pombe. This assay can be used to study both spontaneous breaks arising during DNA replication and induced double-strand breaks created with the S. cerevisiae homothallic endonuclease (HO). The preliminary genetic validation of this assay shows that spontaneous breaks require rad52⁺ but not rad51⁺, while induced breaks require both genes, in agreement with previous studies. This assay will be useful in the field of DNA damage repair for studying mechanisms of intra-chromosomal deletions.

SUBMITTER: Lucas BE

PROVIDER: S-EPMC6789737 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

An Assay to Study Intra-Chromosomal Deletions in Yeast.

Lucas Bailey E BE McPherson Matthew T MT Hawk Tila M TM Wilson Lexia N LN Kroh Jacob M JM Hickman Kyle G KG Fitzgerald Sean R SR Disbennett W Miguel WM Rollins P Daniel PD Hylton Hannah M HM Baseer Mohammed A MA Montgomery Paige N PN Wu Jian-Qiu JQ Petreaca Ruben C RC

Methods and protocols 20190826 3

An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem dir ...[more]

PMID: 31454903

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Project description:Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers.

Dataset Information

An Assay to Study Intra-Chromosomal Deletions in Yeast.

Publications

An Assay to Study Intra-Chromosomal Deletions in Yeast.

Similar Datasets

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