Carrier State ; Gene Expression Profiling ; Hepatitis B ; genetics ; Humans ; Hybridization, Genetic ; Nucleic Acid Hybridization ; methods

We obtained intergenus somatic hybrid between Alhagi pseudalhagi and Astragalus cicer by using electroporation. Agrobacterium rhizogenes A4-transformed A. pseudalhagi protoplasts were treated with iodoacetamide so that they were unable to sustain divisions. A. cicer protoplasts were isolated from a methionine-resistant mutant and did not survive in the medium without plant growth regulator. The intergenus somatic hybrid cells were selected based on physiological complementation between the two parents. We optimized some parameters of electroporation, such as direct current impulse, alternating current impulse and the impulse number. We identified ten hybrid clones by morphological observation, checking the chromosome numbers, isoenzyme analysis and random amplified polymorphic DNA analysis, and obtained regenerated plantlets from three hybrid clones.
Astragalus Plant ; genetics ; physiology ; Electroporation ; Fabaceae ; genetics ; physiology ; Hybridization, Genetic ; Transformation, Genetic

In order to screen microsatellites for conservation genetics studies of the species, a total of 23 microsatellite loci from Korean goral (Naemorhedus caudatus), including 15 previously developed loci and 8 new loci in this study, were tested. Eleven microsatellites were screened and subjected to cross-species amplification using a test panel of four Caprinae species, Japanese serows (Capricornis crispus), Chinese gorals (Naemorhedus goral), Northern chamois (Rupicapra rupicapra) and domestic goats (Capra hircus). In addition, all eleven microsatellites (SY3A, SY12A, SY12B, SY48, SY58, SY71, SY76, SY84, SY84B, SY112, and SY129) satisfied the criteria to be a core set of microsatellites. This core set of microsatellites and cross-species amplification of Korean goral microsatellites were found to be helpful for high-resolution studies for conservation and management of Korean goral and other endangered Caprinae species.
Animals ; Conservation of Natural Resources ; Genetic Variation ; Hybridization, Genetic/*genetics ; Microsatellite Repeats/*genetics ; Republic of Korea ; Ruminants/*genetics

OBJECTIVE: To investigate the clinical phenotype and genetic diagnosis of an infant featuring multiple hair and hyperbilirubinemia. METHODS: Conventional G-banding analysis, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) for the patient were conducted, G-banding analyses of peripheral blood for the infant's parents were also performed. RESULTS: We investigated an infant who carries a unbalanced, maternally inherited karyotype 46, X, der (X) t (X;1) (p11.22; q21.3) in which CMA and FISH analyses disclosed a 1q21.3q44 duplication of 93.03 Mb and Xp22.33p11.22 deletion of 54.53 Mb. CONCLUSION The phenotypes of this infant can probably be attributed to the 1q21.3q44 duplication and Xp22.33p11.22 deletion, which were maternally inherited.
Chromosome Banding ; Chromosome Deletion ; Genetic Testing ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Translocation, Genetic

The cytogenetic analysis of recurring chromosomal aberrations play an important part to decide pathogenesis and prognosis of cancers. However, due to difficulties culturing tumor cells and complexity associated with the lesions, routine cytogenetic studies to analyze chromosomal imbalances are not sufficient. Comparative genomic hybridization (CGH) is a fluorescence in situ hybridization (FISH) technique to identify genomic imbalances in cancers, and array-CGH provides a method to measure the DNA copy-number changes quantitatively at an extremely high resolution and to map them directly onto the complete linear genome sequences. The purpose of this study was to confirm the utility of the CGH and array-CGH in analyzing chromosomal aberrations in lung cancer cell line, NCI-H1373, which was previously analyzed by karyotype analysis. The results of CGH and array-CGH in NCI-H1373 were similar to karyotype analysis. The array-CGH allowed us to pinpoint regions that were gained and lost. In this study, it was confirmed that CGH and array-CGH are an useful screening technique to analyze chromosomal aberrations in tumors.
Cell Line* ; Chromosome Aberrations* ; Comparative Genomic Hybridization ; Cytogenetic Analysis ; Cytogenetics ; DNA ; Fluorescence ; Genome ; Hybridization, Genetic ; In Situ Hybridization ; Karyotype ; Lung Neoplasms* ; Lung* ; Mass Screening ; Prognosis

BACKGROUND AND OBJECTIVES: Cancer of the thyroid is the sixth common cancer in Korea, and fourth common among the Korean women, in particular. Aming the prevalent carcinomas of thyroid, the papillary thyroid carcinoma is the most frequent type. Genomic instability is the characteristic of nearly all tumors as well as thyroid cancers. However, despite the high frequency of papillary thyroid carcinomas, their chromosomal alterations are poorly characterized in Korea. Comparative genomic hybridization (CGH) is a new fluorescence in situ hybridization (FISH) technique to identify genomic imbalances in cancers. In this study, CGH was carried out with the aim of analyzing non-random chromosomal aberrations involved in papillary thyroid carcinomas. MATERIALS AND METHOD: CGH was carried out. Biotin-labeled tumor DNA and digoxigenin-labeled normal DNA were co-hybridized to normal metaphase cells. Then, the ratio of fluorescence was analyzed by an image analyzer. In array-CGH, Cy3 labeled tumor DNA and Cy5 labeled normal DNA were hybridized to microarray template, and then image analysis was performed by microarray image analyzer. RESULTS: Gains of 22q13, 6p24, 7p13, 7q21, 7q31, 8q24, 17q24 and 19p13.3 were found frequently. CONCLUSION: Non-random aberrations which were disclosed in this study might be candidate regions for the abnormal genes involved in papillary thyroid cancer.
Carcinoma, Papillary* ; Chromosome Aberrations* ; Comparative Genomic Hybridization* ; DNA ; Female ; Fluorescence ; Genomic Instability ; Humans ; Hybridization, Genetic ; In Situ Hybridization ; Korea ; Metaphase ; Thyroid Gland* ; Thyroid Neoplasms

The short report will be focused on helping our students to understand commonly used conventional and cutting edge cytogenetic techniques and their clinical applications, the advances and drawbacks of each technique, and how to pick the right test(s) for a specific patient in order to achieve a proper diagnosis efficiently and economically.
Chromosomes ; ultrastructure ; Cytogenetic Analysis ; Cytogenetics ; methods ; Genetic Techniques ; Humans ; In Situ Hybridization, Fluorescence ; Molecular Diagnostic Techniques ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Translocation, Genetic

OBJECTIVETo investigate the efficiency of multi-round fluorescence in situ hybridization (FISH) and its influencing factors in preimplantation genetic diagnosis (PGD).

METHODSA total of 48 couples accepted PGD because of various reasons: 24 with Robertsonian translocations, 16 with reciprocal translocations, 2 with pericentric inversions, one with advanced maternal age who had a previous liveborn of Down syndrome, 3 suffered from sex chromosome abnormalities and 2 repeated spontaneous miscarriages. After 72 retrieval cycles, 432 cleavage stage embryos with more than six cells were biopsied on day three. Only intact nuclei (396) were hybridized in order to verify the chromosomal status of the individual embryos. If previous FISH has failed to give conclusive results while the nuclei remained undamaged, the nuclei were hybridized once again. A total of 870 times of hybridization were conducted to 396 nuclei. Signal identification rates of each round as well as the influence of different probes to the hybridization efficiency were compared. Factors leading to inconclusive FISH results were analyzed as well.

RESULTSFive hundred and thirty five out of 870 hybridizations gave identifiable signals (61.5%). The second and third round FISH showed the best signals with an identification rate of 71.8% and 77.4%, respectively, which were significantly higher than those of the first round (52.8%, P < 0.01), the fourth round (55.8%, P < 0.05, P < 0.01), the fifth round (54.5%, P < 0.05) and the sixth round (27.3%, P < 0.01). The identification rate of centromere specific probe signals (CEP group) was 80.3% and the former three rounds in this group got the best quality of signals with an identification rate of 85.7%, 85.1% and 88.0%, respectively, which was significantly higher than that of the latter three rounds. The identification rate of other probe was much lower than with the CEP probe (55.2% vs. 80.3%, P < 0.01) and the best quality of signal in this group was achieved in the fifth round (72.7%), followed by the second round (66.1%) and the third round (63.8%). The identification rate of the first round (50.3%) and the sixth round (22.2%) were significantly lower compared with the second round (P < 0.01). During the 6 rounds of FISH, 335 hybridizations did not give conclusion results (38.5%, 335/870). The main cause of unidentification was weak signals (20.9%, 182/870). Other common factors included background interference (7.6%, 66/870) and failed hybridization (6.1%, 53/870). Rare causes included nucleus damage (1.8%, 16/870), nucleus loss (1.1%, 10/870) and signal split/overlap (0.9%, 8/870).

CONCLUSIONMulti-round FISH can improve the utility of single nucleus in PGD and the former three rounds have the highest efficiency. The hybridization effect of CEP is better than other probe. Poor signal quality is the common cause of unidentification results.

Female ; Genetic Testing ; methods ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Male ; Pregnancy ; Preimplantation Diagnosis ; methods ; Prenatal Diagnosis ; Translocation, Genetic

OBJECTIVE: To explore the effect of chromosomal translocations on the composition of embryonic chromosomes and its mechanism. METHODS: For 52 couples with one partner carrying a chromosomal translocation, results of next generation sequencing of all embryos derived from 61 cycles were divided into different groups based on the type of translocations, gender of the carrier, and maternal age. Effect of parental chromosomal translocations on the composition of embryonic chromosomes of each group was analyzed. RESULTS: A significant difference was found between carriers of reciprocal and Robertsonian translocations in terms of proportion of abnormal embryos and structurally normal chromosomes (63.3% vs. 27.5%, and 1.1% vs. 0.3%, respectively). Compared with male carriers, there was an increase in the rate of abnormalities for female carriers (67.2% vs. 58.3% for reciprocal translocations, and 45.5% vs. 13.8% for Robertsonian translocations). The risk for chromosomal abnormality also increased with the maternal age. No significant difference was found in the proportion of abnormal embryos between carriers divided by involvement of acrocentric chromosomes or terminal chromosomal breakpoints. CONCLUSION The types of parental translocation, gender of carrier, maternal age, and interchromosomal effect have certain effect on the composition of embryonic chromosomes.
Chromosomes, Human ; genetics ; Female ; Genetic Carrier Screening ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Maternal Age ; Pregnancy ; Preimplantation Diagnosis ; Translocation, Genetic

OBJECTIVETo investigate the genetic diversity and structure of Dendrobium huoshanense, a (CT)n enriched microsatellite library was constructed using a magnetic beads enrichment procedure.

METHODThe 3'-biotinylated oligonucleotide probe was used to hybridize with the digested D. huoshanense genomic DNA fragments whose both ends were ligated with adaptors. The hybridized complex was then combined with the streptavidin-coated magnetic beads. The captured microsatellite fragments were eluted, collected and cloned into pMD19-T vector. The recombinant plasmids were transformed into Escherichia coli DH5alpha competent cells. The clones that yielded two or more bands contained microsatellite fractions. Positive clones were screened and sequenced. Thirty pairs of primers were designed and synthesized. Polymorphism at each locus was determined using 24 individuals from a natural population from Huoshan county town in Anhui province.

RESULTTwelve polymorphic microsatellite loci from the microsatellite-enriched genomic library were newly developed across 24 D. huoshanense individuals. In total, 65 alleles were identified, and the number of alleles per locus ranged from 2 to 8. The mean observed and expected heterozygosities were 0.500 and 0.638, respectively. Two loci significantly deviated from Hardy-Weinberg equilibrium (P<0.05), which could be due to the presence of null alleles. Furthermore, three of twelve loci showed significant linkage disequilibrium (P<0.05).

CONCLUSIONThese results suggest that the identified polymorphic microsatellite markers will be useful in population genetic studies of D. huoshanense.

Dendrobium ; genetics ; Genomic Library ; Microsatellite Repeats ; Molecular Sequence Data ; Nucleic Acid Hybridization ; Polymorphism, Genetic