Molecular Karyotyping Research Articles

Improved molecular karyotyping in glioblastoma

Uneven replication creates artifacts during whole genome amplification (WGA) that confound molecular karyotype assignment in single cells. Here, we present an improved WGA recipe that increased coverage and detection of copy number variants (CNVs) in single cells. We examined serial resections of glioblastoma (GBM) tumor from the same patient and found low-abundance clones containing CNVs in clinically relevant loci that were not observable using bulk DNA sequencing. We discovered extensive genomic variability in this class of tumor and provide a practical approach for investigating somatic mosaicism.

Clinical application of targeted next-generation sequencing in fetuses with congenital heart defect.

To assess the value of targeted next-generation sequencing (NGS) in prenatal diagnosis of congenital heart defects (CHD) and to investigate the genetic etiology of prenatal CHD. Forty-four fetuses with CHD, normal molecular karyotype and negative chromosomal microarray results underwent targeted NGS. Fetal genomic DNA was extracted directly from amniotic fluid cells in each prenatal case. A customized targeted-NGS panel of 77 CHD-associated genes was designed to detect variants in the coding regions and the splicing sites of these genes. The detected variants were classified as pathogenic, likely pathogenic, of uncertain significance, likely benign or benign, following the guidelines recommended by the American College of Medical Genetics and Genomics. The detection rates of targeted NGS for pathogenic and likely pathogenic variations were 13.6% (6/44) and 2.3% (1/44), respectively. The turnaround time of the test was 3 weeks. The six pathogenic variations were identified on the genes CHD7 (CHARGE syndrome), CITED2 (tetralogy of Fallot, ventricular septal defect and atrial septal defect), ZFPM2 (tetralogy of Fallot), MYH6 (atrial septal defect, familial isolated dilated cardiomyopathy) and, in two cases, KMT2D (Kabuki syndrome). The likely pathogenic variation was detected on JAG1, which is associated with tetralogy of Fallot and Alagille syndrome. Sanger sequencing in the fetuses and their parents indicated that all seven mutations were de novo. Variations of uncertain significance were detected in 79.5% of cases. Targeted NGS in fetuses with isolated and non-isolated CHD achieved a high diagnostic yield in our cohort, with an acceptable turnaround time for the prenatal setting. Our results have important implications for clinical management and genetic counseling. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Preimplantation genetic testing of Robertsonian translocation by SNP array-based preimplantation genetic haplotyping.

The present study attempted to confirm a method that distinguishes a balanced Robertsonian translocation carrier embryo from a truly normal embryo in parallel with comprehensive chromosome screening (CCS). Comprehensive chromosome screening was performed in 107 embryos from 11 couples carrying Robertsonian translocations. Among them, embryos from 2 families had been transferred before the diagnosis of translocation, which resulted in successful pregnancies; embryos from the remaining families were transferred after the identification of translocations. The single nucleotide polymorphism (SNP) genotypes were acquired on a genome-wide basis, and breakpoint regions and flanking were assessed by establishing haplotypes. The predicted karyotypes from the transferred embryos were confirmed by prenatal diagnosis. Among the 9 families finally undergoing translocation diagnosis, the amniotic cell karyotypes of 3 families were concordant with the results predicted by preimplantation genetic haplotyping, revealing a good consistency rate. After CCS, the euploid embryos from 2 other families could not be further detected because of the absence of abnormal embryos as probands. Molecular karyotypes and haplotypes could be established with SNP microarray simultaneously in each embryo. SNP array-based PGT can simultaneously complete the CCS and identify Robertsonian translocation carriers, thus making it possible to prevent Robertsonian translocations from being passed to subsequent generations.

Prospective chromosome analysis of 3429 amniocentesis samples in China using copy number variation sequencing

Next-generation sequencing is emerging as a viable alternative to chromosome microarray analysis for the diagnosis of chromosome disease syndromes. One next-generation sequencing methodology, copy number variation sequencing, has been shown to deliver high reliability, accuracy, and reproducibility for detection of fetal copy number variations in prenatal samples. However, its clinical utility as a first-tier diagnostic method has yet to be demonstrated in a large cohort of pregnant women referred for fetal chromosome testing. We sought to evaluate copy number variation sequencing as a first-tier diagnostic method for detection of fetal chromosome anomalies in a general population of pregnant women with high-risk prenatal indications. This was a prospective analysis of 3429 pregnant women referred for amniocentesis and fetal chromosome testing for different risk indications, including advanced maternal age, high-risk maternal serum screening, and positivity for an ultrasound soft marker. Amniocentesis was performed by standard procedures. Amniocyte DNA was analyzed by copy number variation sequencing with a chromosome resolution of 0.1 Mb. Fetal chromosome anomalies including whole chromosome aneuploidy and segmental imbalances were independently confirmed by gold standard cytogenetic and molecular methods and their pathogenicity determined following guidelines of the American College of Medical Genetics for sequence variants. Clear interpretable copy number variation sequencing results were obtained for all 3429 amniocentesis samples. Copy number variation sequencing identified 3293 samples (96%) with a normal molecular karyotype and 136 samples (4%) with an altered molecular karyotype. A total of 146 fetal chromosome anomalies were detected, comprising 46 whole chromosome aneuploidies (pathogenic), 29 submicroscopic microdeletions/microduplications with known or suspected associations with chromosome disease syndromes (pathogenic), 22 other microdeletions/microduplications (likely pathogenic), and 49 variants of uncertain significance. Overall, the cumulative frequency of pathogenic/likely pathogenic and variants of uncertain significance chromosome anomalies in the patient cohort was 2.83% and 1.43%, respectively. In the 3 high-risk advanced maternal age, high-risk maternal serum screening, and ultrasound soft marker groups, the most common whole chromosome aneuploidy detected was trisomy 21, followed by sex chromosome aneuploidies, trisomy 18, and trisomy 13. Across all clinical indications, there was a similar incidence of submicroscopic copy number variations, with approximately equal proportions of pathogenic/likely pathogenic and variants of uncertain significance copy number variations. If karyotyping had been used as an alternate cytogenetics detection method, copy number variation sequencing would have returned a 1% higher yield of pathogenic or likely pathogenic copy number variations. In a large prospective clinical study, copy number variation sequencing delivered high reliability and accuracy for identifying clinically significant fetal anomalies in prenatal samples. Based on key performance criteria, copy number variation sequencing appears to be a well-suited methodology for first-tier diagnosis of pregnant women in the general population at risk of having a suspected fetal chromosome abnormality.

Prenatal diagnosis of Wolf-Hirschhorn syndrome: Ultrasonography and molecular karyotyping results

ObjectiveTo present the experience on prenatal diagnosis of Wolf-Hirschhorn syndrome (WHS) to further delineate the fetal presentation of this syndrome. Study designThis was a retrospective analysis of ten pregnancies with fetal WHS identified by chromosomal microarray (CMA). Clinical data were reviewed for these cases, including maternal demographics, indications for invasive testing, sonographic findings, CMA results and pregnancy outcomes. ResultsThree cases were diagnosed at the first trimester because of an increased NT or cystic hygroma. The remaining seven cases were identified at late gestation for abnormal ultrasound findings. CMA revealed 4p deletions to be terminal in all of the ten cases. Deletion sizes ranged from 2.05 to 19.02 Mb. ConclusionPrenatal findings such as increased NT, severe and early onset intrauterine growth retardation, and renal dysplasia or oligohydramnios should warrant the diagnosis of WHS and invasive testing using CMA.

Genetic investigation of 93 families with microphthalmia or posterior microphthalmos.

Microphthalmia is a developmental eye defect that is highly variable in severity and in its potential for systemic association. Despite the discovery of many disease genes in microphthalmia, at least 50% of patients remain undiagnosed genetically. Here, we describe a cohort of 147 patients (93 families) from our highly consanguineous population with various forms of microphthalmia (including the distinct entity of posterior microphthalmos) that were investigated using a next-generation sequencing multi-gene panel (i-panel) as well as whole exome sequencing and molecular karyotyping. A potentially causal mutation was identified in the majority of the cohort with microphthalmia (61%) and posterior microphthalmos (82%). The identified mutations (55 point mutations, 15 of which are novel) spanned 24 known disease genes, some of which have not or only very rarely been linked to microphthalmia (PAX6, SLC18A2, DSC3 and CNKSR1). Our study has also identified interesting candidate variants in 2 genes that have not been linked to human diseases (MYO10 and ZNF219), which we present here as novel candidates for microphthalmia. In addition to revealing novel phenotypic aspects of microphthalmia, this study expands its allelic and locus heterogeneity and highlights the need for expanded testing of patients with this condition.

A rapid and reliable chromosome analysis method for products of conception using interphase nuclei.

BackgroundKaryotype determination has a central role in the genetic workup of pregnancy loss, as aneuploidy (trisomy and monosomy) and polyploidy (triploidy and tetraploidy) are the cause in at least 50% of first trimester, 25% of second trimester, and 11% of third trimester miscarriages. There are several limitations with the current approaches of obtaining a karyotype using traditional cytogenetics, fluorescence in situ hybridization with a limited number of probes, and chromosomal microarray. These include culture failure, incomplete results, lower sensitivity, and longer reporting time.MethodsTo overcome current limitations, a novel molecular assay is developed with a Standard Resolution Interphase Chromosome Profiling probe set which is a variation of the recently developed High Resolution probe set. It generates a molecular karyotype that can detect all major changes commonly associated with pregnancy loss. Initial familiarization of signal patterns from the probe set was used, followed by validation of the method using 83 samples from miscarriages in a blind study from three different laboratories. Finally, the clinical utility of the method was tested on 291 clinical samples in two commercial reference laboratory settings on two different continents.ResultsThe new molecular approach not only identified all the chromosome changes observed by current methods, but also significantly improved abnormality detection by characterizing derivative chromosomes and finding subtle subtelomeric rearrangements, balanced and unbalanced. All Robertsonian translocations were also detected. The abnormality rate was 54% on clinical samples from commercial laboratory 1 and 63% from laboratory 2.ConclusionThe attributes of this method make it an ideal choice for the genetic workup of miscarriages, namely (1) near 100% successful results, (2) greater sensitivity than conventional chromosome analysis or FISH panels, (3) rapid reporting time, and (4) favorable comparisons with chromosomal microarray.

Diagnostic efficacy and new variants in isolated and complex autism spectrum disorder using molecular karyotyping.

Autism spectrum disorder (ASD) is a group of the neurodevelopment disorders presenting as an isolated ASD or more complex forms, where a broader clinical phenotype comprised of developmental delay and intellectual disability is present. Both the isolated and complex forms have a significant causal genetic component and submicroscopic genomic copy number variations (CNV) are the most common identifiable genetic factor in these patients. The data on microarray testing in ASD cohorts are still accumulating and novel loci are often identified; therefore, we aimed to evaluate the diagnostic efficacy of the method and the relevance of implementing it into routine genetic testing in ASD patients. A genome-wide CNV analysis using the Agilent microarrays was performed in a group of 150 individuals with an isolated or complex ASD. Altogether, 11 (7.3%) pathogenic CNVs and 15 (10.0%) variants of unknown significance (VOUS) were identified, with the highest proportion of pathogenic CNVs in the subgroup of the complex ASD patients (14.3%). An interesting case of previously unreported partial UPF3B gene deletion was identified among the pathogenic CNVs. Among the CNVs with unknown significance, four VOUS involved genes with possible correlation to ASD, namely genes SNTG2, PARK2, CADPS2 and NLGN4X. The diagnostic efficacy of aCGH in our cohort was comparable with those of the previously reported and identified an important proportion of genetic ASD cases. Despite the continuum of published studies on the CNV testing in ASD cohorts, a considerable number of VOUS CNVs is still being identified, namely 10.0% in our study.

Parental origin of deletions and duplications \u2013 about the necessity to check for cryptic inversions

BackgroundCopy number variants (CNVs) are the genetic bases for microdeletion/ microduplication syndromes (MMSs). Couples with an affected child and desire to have further children are routinely tested for a potential parental origin of a specific CNV either by molecular karyotyping or by two color fluorescence in situ hybridization (FISH), yet. In the latter case a critical region probe (CRP) is combined with a control probe for identification of the chromosome in question. However, CNVs can arise also due to other reasons, like a recombination-event based on a submicroscopic, cryptic inversion in one of the parents.ResultsSeventy-four patients with different MMSs and overall 81 CNVs were studied here by a novel three color FISH approach. The way how three locus-specific probes are selected (one is the CRP and two are flanking it in a distance of 5-10 Mb) enables to detect or exclude two possible parental conditions as origins of the CNV seen in the index: (i) direct parental origin of the CNV (deletion or duplication) or (ii) a parental cryptic inversion. Thus, for overall 51/81 CNVs (63%) a parental origin could be determined. 36/51 (70.5%) inherited the CNV directly from one of the parents, but 15/51 (29.5%) were due to an exclusively by three color FISH detectable parental inversion. A 2:1 ratio of maternal versus paternal inheritance was found. Also almost two times more male than female were among the index patients.ConclusionThe new, here suggested three color FISH approach is suited for more comprehensive parental studies of patients with MMS. The detection rate for parental origin was increased by 140% in this study. Still, for 30/81 cases (37%) no reason for the ‘de novo’ MMS in the affected index patient could be found by the here suggested FISH-probe set.

Molecular karyotyping in early miscarriages: potential for the routine use of cytogenetic microarrays

Approximately 15% of all the pregnancies end with early miscarriage, with approximately half of these cases resulting from chromosomal abnormalities of the foetus (Simpson and Bombard 1987). The cu...

88 - Molecular Karyotyping in Children and Adolescents with Gender Dysphoria

Purpose: The presence of a disorder of sexual development (DSD) acts as a diagnostic specifier for gender dysphoria (GD) under DSM-5, while the ICD-10 specifically states that its equivalent diagnosis, Gender Identity Disorder (GID), must not be the result of a chromosomal abnormality. For these reasons, routine karyotyping has been previously advocated in the clinical work-up of children and adolescents with suspected GD or GID. However, the utility of such testing remains unclear.

Application of Molecular Karyotyping in Acute Myeloid Leukemia: A Review

Acute myeloid leukemia (AML) is an aggressive disease characterized by the overproduction of immature myeloid cells that accumulate in blood and bone marrow. Integration of genetic findings and clinicopathological information is crucial in establishing the diagnosis, prognosis and determining the therapeutic approach in the management of AML patients. In recent years, the AML classification has evolved from morphology to cytogenetics/molecular genetics-based findings, which is essential in the detection of chromosomal abnormalities and has provided the framework for the diagnosis and risk-stratification in AML. Moreover, with advances in molecular karyotyping such as comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays, various limitations of conventional diagnostic approaches have been overcome. Hence, this review focuses on the insights into molecular karyotyping using CGH and SNP arrays which enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. Technical hindrances of these methods (e.g. regions of losses, gains, or “undulating waves”) are also discussed in the context of AML.

Genetic counseling prior to Assisted Reproductive Technology procedures in the era of cytogenomics.

The possibility of sequencing hundreds of genes simultaneously and performing molecular karyotyping thanks to the introduction of novel genetic tools has expanded the use of preconception screening for blastocyst recessive mutations and aneuploidies before embryo transfer, with the ultimate purpose of increasing the proportion of normal healthy newborns. Since medically-assisted reproduction procedures are increasingly required to be eugenic, and the aforementioned genetic tests cover only half of the potential genetic diseases occurring at birth, it seems reasonable to incorporate genetic counseling in the practice of assisted reproduction to avoid prosecution for malpractice.

Integrated Karyotyping of Woodland Strawberry (Fragaria vesca) with Oligopaint FISH Probes

Chromosome identification is critical for many aspects of cytogenetic research. However, for Fragaria vesca, definite identification of individual chromosomes is almost impossible because of their small size and high similarity. Here, we demonstrate that bulked oligonucleotide (oligo) probes can be used as chromosome-specific DNA markers for chromosome identification in F. vesca. Oligos specific to entire pseudochromosomes in the draft genome of F. vesca were identified and synthesized as libraries. In all, we synthesized 6 oligo libraries corresponding to 6 pseudochromosomes of F. vesca. These libraries were amplified and labeled as probes for fluorescence in situ hybridization (FISH). Two rounds of multicolor FISH analysis were sequentially conducted on the same metaphase cells with each round including 3 probe libraries, which permitted simultaneous identification of all chromosomes of F. vesca. Moreover, 45S and 5S rDNA were mapped to chromosomes 1, 2, and 7, respectively. A karyotype of metaphase chromosomes was constructed, representing the first FISH-based molecular cytogenetic karyotype of F. vesca. Our study can serve as a basis for future comparative cytogenetic research through cross-species chromosome painting using bulked oligo probes and will facilitate the application of breeding technologies that rely on the identification of chromosomes in the genus Fragaria.

SVAtools for junction detection of genome-wide chromosomal rearrangements by mate-pair sequencing (MPseq)

Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10 kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions and gene fusions, and characterize the genomic structure of complex rearrangements. To illustrate the power of SVAtools' junction detection methods to provide comprehensive molecular karyotypes, MPseq data were compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, FISH and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools' junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.

The establishment and application of preimplantation genetic haplotyping in embryo diagnosis for reciprocal and Robertsonian translocation carriers

BackgroundPreimplantation genetic diagnosis (PGD) is now widely used to select embryos free of chromosomal copy number variations (CNV) from chromosome balanced translocation carriers. However, it remains a difficulty to distinguish in embryos between balanced and structurally normal chromosomes efficiently.MethodsFor this purpose, genome wide preimplantation genetic haplotyping (PGH) analysis was utilized based on single nucleotide polymorphism (SNP) microarray. SNPs that are heterozygous in the carrier and, homozygous in the carrier’s partner and carrier’s family member are defined as informative SNPs. The haplotypes including the breakpoint regions, the whole chromosomes involved in the translocation and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference and embryos. In order to perform this analysis, a reference either a translocation carrier’s family member or one unbalanced embryo is required. The positions of translocation breakpoints are identified by molecular karyotypes of unbalanced embryos. The recombination of breakpoint regions in embryos could be identified.ResultsEleven translocation families were enrolled. 68 blastocysts were analyzed, in which 42 were unbalanced or aneuploid and the other 26 were balanced or normal chromosomes. Thirteen embryos were transferred back to patients. Prenatal cytogenetic analysis of amniotic fluid cells was performed. The results predicted by PGH and karyotypes were totally consistent.ConclusionsWith the successful clinical application, we demonstrate that PGH was a simple, efficient, and popularized method to distinguish between balanced and structurally normal chromosome embryos.

Eosinophilic Solid and Cystic Renal Cell Carcinoma (ESC RCC): Further Morphologic and Molecular Characterization of ESC RCC as a Distinct Entity.

Eosinophilic solid and cystic renal cell carcinoma (ESC RCC) has been recently described as a unique and indolent renal neoplasm, found in female patients with and without tuberous sclerosis complex. Although ESC RCC has a distinct morphology and frequent CK20 reactivity, its molecular karyotype has been previously studied only in few cases. We identified 19 ESC RCC from multiple institutions; all patients were female individuals without clinical features of tuberous sclerosis complex. Molecular karyotyping was performed in 13 cases (12 with informative result). The median age was 55 years (range: 32 to 79 y). The tumors were yellow-gray with a median size of 31 mm (range: 12 to 135 mm) and showed solid and cystic gross appearance. All tumors demonstrated typical microscopic features with solid areas admixed with variably sized macrocysts and microcysts. The cells showed eosinophilic cytoplasm with granular cytoplasmic stippling and round-to-oval nuclei. CK20 was positive in 14/19 (74%) cases. Stage pT1 was found in 17/19 (89%) patients (pT1a in 12, pT1b in 5); 1 patient each had pT2a and pT3a. A total of 15/16 patients with available follow-up were alive and without evidence of disease progression, after 1 to 169 months (median: 44 mo; mean: 49.6 mo); 3 died of other causes. The most common copy number gains were 16p13.3-16q23.1 (33% to 67%), 7p21.2-7q36.2 (42% to 50%), 13q14.2 (33%), and 19p12 (33%). The most common copy number losses included Xp11.21 (42%) and 22q11.23 (33%). Loss of heterozygosity was most frequently found at 16p11.2-11.1 (75%), Xq11.1-13.1 (75%), Xq13.1-21.1 (33%), 11p11.2-11.11 (33%), 9q21.1-22.2 (33%), and 9q33.1 (33%). ESC RCC demonstrates common molecular karyotype alterations, which further support its distinct nature.

Biology of B. sorokiniana (syn. Cochliobolus sativus) in genomics era

Bipolaris sorokiniana (Sacc.) Shoemaker is a hemi-biotrophic fungal pathogen, which is an anamorph (teleomorph Cochlibolus sativus). It causes spot blotch, root rot and leaf spot diseases in a number of cereals including wheat, barley and other small grain cereals. In the genomics era, the fungus has been subjected to a variety of studies using molecular approaches. Correct chromosome number was determined and molecular karyotypes were prepared using contour-clamped homogeneous electric field. Molecular maps were prepared using markers like RFLPs, SSRs, RAPDs and SNPs. For this purpose, segregating progenies derived from crosses between diverse isolates of the pathogen were used. Whole genome sequencing (WGS) data was collected not only for B. sorokiniana isolates, but also for several species of Cochliobolus. Genes involved in secondary metabolism and virulence were identified from genome sequences. The WGS data has also been utilized for comparative genomics giving useful information about evolutionary trends. A brief account of this information is presented in this review.

Genetic screening by sequencing technology to predict embryo developmental competence of the male gamete

We carried out a whole molecular karyotype with gene copy number variant (CNV) assessment to properly profile the male genome. Whole genome assessment was carried out on spermatozoa of men undergoing ART by ICSI and subdivided according to their ability to carry on a successful pregnancy and compared to those with an early miscarriage. Ejaculates were obtained from consenting men and 2 donor specimens. FISH was performed on at least 1000 sperm cells with a threshold of >1.6%. DNA was extracted from ∼500 spermatozoa per sample followed by PCR-based random hexamer amplification, yielding an average DNA concentration of 580±348ng/ul and adequate for Next Generation Sequencing (NGS). CNVs were calculated by CASAVA and VarScan2. The average male and female age was 38.3±7yrs and 37.5±5yrs, respectively. Six couples in 15 ICSI cycles achieved a clinical pregnancy rate of 80%, defining the fertile group. Ten couples treated in 21 cycles achieved a pregnancy rate of only 23.8% that, however, resulted in a first trimester pregnancy loss defining the infertile group. For those 2 cohorts, sperm aneuploidy by FISH was comparable at 2.8% and 3.1%, respectively. Whole genome sequencing, on the other hand, yielded an overall aneuploidy of 4.0% for the fertile and 8.6% for the infertile group (P<0.00001). Gene duplications were 2.3% in the control, progressively increased to 8.4% in the fertile cohort, and reached 95% in the infertile (P<0.00001). Similarly, gene sequencing deletions in the control at 4.4% rose to 6.1% in the fertile and all the way to 92.5% in the infertile cohorts (P<0.00001). Interestingly, for the fertile and infertile cohorts there was a remarkable imbalance of DPY19L2 and ADAM3A, responsible for acrosomal development and sperm-egg fusion, respectively. On the other hand, in the infertile men, we identified additional genes such as HAUS1, KIF4A, XRN1, and NLRP7, involved in centrosome integrity, spindle stabilization, telomere maintenance, and trophoblast proliferation, respectively. In infertile couples where standard tests fail to provide information on male gamete competence, DNA sequencing provides a comprehensive method for genetic assessment. Detection of CNV identifies specific gene imbalances that may guide towards the appropriate ART procedure as well as predict a successful progression of pregnancy.

Using Pulsed-Field Gel Electrophoresis to Analyze Schizosaccharomyces pombe Chromosomes and Chromosomal Elements.

Pulsed field gel electrophoresis (PFGE) uses alternatively oriented pulsed electrical fields to separate large DNA molecules. Here, we describe PFGE protocols and conditions for separating and visualizing chromosomes between 0.5 and 6 Mb (optimal for analyzing the endogenous fission yeast chromosomes of 5.7, 4.6, and 3.5 Mb), and for shorter chromosomal elements of between 50 and 600 kb, such as the 530 kb Ch16 minichromosome. In addition to determining chromosome size, this technique has a wide range of applications, including determining whether DNA replication or repair is complete, defining the molecular karyotype of cells, analyzing chromosomal rearrangements, assigning genes or constructs to particular chromosomes, and isolating DNA from specific chromosomes.