inter-SSR PCR Research Articles

Genomic Instability in the Progression of Sporadic Nasopharyngeal Carcinoma

Genomic instability reflecting the susceptibility of the genome to acquire multiple genetic alterations plays a major role in tumorigenesis and tumor progression. We evaluated the prognostic significance of the extent of genomic instability in nasopharyngeal carcinoma. Genomic instability was assessed by inter-simple sequence repeats polymerase chain reaction (inter-SSR PCR) in 38 patients with nasopharyngeal carcinoma. Characterization and verification of band alterations shared in different tumors were carried out by sequencing and nest PCR. 31 (81.6%) of 38 patients showed genomic alterations, and genomic instability index ranged from 0 to 16.2%. A gain-based genomic damage shared in 6 tumors was identified on chromosome 6q27, a new mutator phenotype in nasopharyngeal carcinoma. Significantly more genomic alteration was found in patients without 5-year survival than that with 5-year survival (P<0.05), suggesting that higher genomic instability predicts a poor prognosis in nasopharyngeal carcinoma. Our data suggests that genomic instability can be an early event marker in carcinogenesis of nasopharyngeal carcinoma. Also, aggravation of genomic alterations is a poor prognosis for cancer recovery.

Genomic instability in invasive breast carcinoma measured by inter-Simple Sequence Repeat PCR

We have measured genomic instability in invasive breast carcinomas and assessed the relationship of genomic instability to known tumor prognostic factors. DNAs from tumors and adjacent normal tissue of 18 breast cancer patients were subjected to inter-Simple Sequence Repeat (inter-SSR) PCR for quantitation of tumor genomic instability. Associations between genomic instability level and known breast cancer prognostic factors were evaluated using the Pearson Product Moment Correlation, the Kruskal-Wallis test of independent samples and the Mann-Whitney non-parametric test. Genomic instability was detected by inter-SSR PCR in over 90% of the breast tumors. The mean instability index was 3.08% (0-7.59%), approximately the same mean value observed in studies of colorectal and thyroid carcinomas. Significantly higher levels of instability were associated with tumors exhibiting necrosis. Genomic instability as measured is detected in the majority of breast cancers at levels comparable to other tumor types. Hypoxia, such as that observed in necrotic regions of tumors, has been associated with elevated genomic damage. We hypothesize that the higher levels of genomic instability detected in necrotic tumors is a consequence of hypoxia-associated DNA damage.

Genomic heterogeneity and instability in colorectal cancer: spectral karyotyping, glutathione transferase-Ml and ras

Genomic instability in cancer is frequently described as being either chromosomal instability or microsatellite instability, although when events within chromosomes are monitored, extensive intrachromosomal instability is also found. Spectral karyotyping was used to visualize how extensively genomic instability gives rise to intratumor genomic heterogeneity in sporadic colorectal carcinomas. Two factors were then examined which might relate to intrachromosomal instability in colorectal cancers: the presence of the glutathione transferase-Ml gene to detoxify potential carcinogens, and the presence of activated ras which has been associated with chromosomal instability when first expressed. Intrachromosomal genomic instability was previously determined by inter-(simple sequence repeat) PCR (inter-SSR PCR) and by fractional allelic loss rate for 348 markers. GSTM1 status was determined for each of 49 tumors through use of specific PCR, and 28 of the tumors showed the GSTM1 null genotype. A significant association was found between GSTMl-null status and elevated inter-(simple sequence repeat) PCR instability. In contrast, no association was found with fractional allelic loss rate. The first exons of the K-ras and H-ras oncogenes were sequenced in 72 colorectal cancers; 19 of the tumors had a mutation in codon 12 of the K-ras gene (24.5%), but no H-ras mutations were found. A weak correlation ( p = 0.10) was observed between mutant K-ras and inter-(simple sequence repeat) PCR genomic instability, and no association existed with fractional allelic loss rate.

Genomic instability in oral squamous cell carcinoma: relationship to betel-quid chewing

Genomic instability in repeated DNA sequences is exhibited by a variety of cancer types, including oral squamous cell carcinoma. Exposure to carcinogenic compounds may further increase the instability. We have used Inter-Simple Sequence Repeat (Inter-SSR) PCR methodology to detect genetic alterations in 37 oral cancer patients who had chewed betel-quid. Thirty-eight percent of DNA from tumors had genomic alterations in the sequences flanked by (CA) 8 and (GT) 8 repeats. Patients with tumor DNAs harboring genomic alterations had a two-fold higher consumption of betel-quid than patients without alterations in tumor DNA. Matched normal and tumor DNAs were also screened for microsatellite instability where four patients (10.8%) showed alterations in at least one microsatellite marker but there was no relationship between this phenotype and betel-quid chewing. These data indicate that exposure to carcinogens present in the betel-quid may contribute to genomic instability detected by inter-SSR PCR in a subset of oral tumors.

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Assessment of genetic diversity of tea (Camellia sinensis (L.) O. Kuntze) by inter-simple sequence repeat polymerase chain reaction

Twenty-five diverse tea (Camellia sinensis(L.) O. Kuntze) cultivars were analyzed using the simple sequence repeat anchored polymorearse chain reaction (SSR-anchored PCR) or Inter SSR-PCR (ISSR). Out of the 45 primers 12were chosen for final study. These amplified a total of 130 bands out of which108 (84%) were polymorphic. A dendrogram was constructed using UPGMA method revealed three distinct clusters of Cambod, Assamand China type, which concur with the known taxonomical classification of tea. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting and molecular taxonomic classification of tea genotypes.

IDENTIFICATION OF POTATO VARIETIES BY DNA PROFILING

Two DNA profiling methods have been used for variety discrimination in potatoes. Sequence tagged site analysis of microsatellites was shown to be very efficient and was more reliable than inter-SSR PCR analysis. Using only 3 microsatellite primer pairs, it was possible to distinguish between 50 potato varieties.

5'-Anchored simple-sequence repeat primers are useful for analysing potato somatic hybrids.

Somatic hybrids between Solanum tuberosum cv 'Brodick' and S. sanctae-rosae were selected for having elevated chromosome numbers, intermediate morphology and potato cyst nematode resistance (derived from S. sanctae-rosae). DNA was extracted from this material and subjected to inter-SSR PCR directed by oligonucleotide primers comprising 5'-anchored di- and tri-nucleotide repeat motifs. PCR products were separated by polyacrylamide gel electrophoresis and visualised by silver staining. An average of 30.31 bands per primer were generated in each parent and, on average, 8.19 of these bands were polymorphic. S. sanctae-rosae-specific bands were observed in the hybrid material as was evidence of deletions, substitutions and rearrangements. The relative merits of this technique for the analysis of somatic hybrid material are discussed.

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3'-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5′-anchored and two 3′-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5′-anchored primers revealed more distinct polymorphic markers than the 3′-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

Evaluation of genetic diversity and genome fingerprinting ofPandorea(Bignoniaceae) by RAPD and inter-SSR PCR

Evaluation of the genetic relationship between five Pandorea jasminoides and eight Pandorea pandorana cultivars was investigated using random amplification of polymorphic DNA and inter-SSR (simple sequence repeat) PCR. Twenty-five random primers generated 375 polymorphic products with a mean number of 15 polymorphic bands per primer. The mean number of polymorphic products obtained by inter-SSR PCR was 18.7 per primer. Random amplification of polymorphic DNA and inter-SSR amplification of genomic DNA revealed higher genetic variability among the cultivars of P. pandorana than among those of P. jasminoides. Two random primers each produced species-specific amplification products in both the Pandorea species, while four random primers and two SSRs produced species-specific amplification products in only one of the species. A distinct cultivar-specific DNA fingerprint for each of the 13 cultivars was obtained by 2 random primers and 1 SSR primer. A dendrogram constructed on the basis of 513 amplification products generated by random amplification of polymorphic DNA and inter-SSR PCR showed the separation of 13 cultivars into two major groups that corresponded to P. jasminoides and P. pandorana. The data resulting from random amplification of polymorphic DNA and inter-SSR PCR were congruent. Our study showed that it should be possible to take advantage of existing genetic variability of P. pandorana to enhance the narrow genetic base of P. jasminoides by interspecific crosses. Such crosses would have the potential to generate novel commercially important elite cultivars of Pandorea.Key words: Pandorea, RAPD, inter-SSR, DNA fingerprint, genetic variability.

Evaluation of genetic diversity and genome fingerprinting of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR

Evaluation of genetic diversity and genome fingerprinting of <i>Pandorea</i> (Bignoniaceae) by RAPD and inter-SSR PCR

Efficiency of Inter-Simple Sequence Repeat PCR for Detecting Somaclonal Variation among Leaf-Culture-Regenerated Plants of Horseradish.

Regenerated plants were obtained by culturing leaf explants of Akame and Aome strains of horseradish (Armoracia rusticana Gaertn.) on MS agar-solidified medium supplemented with 1 mg/l NAA + 1 mg/l TDZ. Extracted DNA from leaves of mother plants was amplified using random and simple sequence repeat (SSR) primers. The SSR primers generated a larger nwnber of scorable bands than the random ones, although inter-and intra-strain polymorphism in the banding pattern was detected by both SSR and random primers at alnrost the same level. An SSR primer, (CAGA)4, was selected for detecting somaclonal variation among regenerants from leaf-derived calli. Somaclonal variation among the regenerants was observed in the PCR products as well as in the leaf shape and color. Seventy one percent of the regenerants exhibited different banding patterns from those of their mother plant, whereas variation in the leaf shape and color was observed in 5 % of the regenerants. These findings suggest that inter-SSR PCR is an efficient tool for detecting somaclonal variation in horseradish. The frequency of the regenerants exhibiting different banding patterns from those of their mother plants and the number of variant bands per regenerant were higher in the Akame than in the Aome strain, suggesting that somaclonal variation in horseradish was affected by the genotype. Fifty percent and 60% of the missing and newly acquired bands, respectively, for the same fragment size were common to the regenerants originating from different leaf-derived calli, suggesting that there were mutable sites in the horseradish genome during the tissue culture process.

PCR markers distinguish Plantago major subspecies

Plantago major plants from several Scottish and Dutch locations were surveyed for their genetic variation using PCR markers, namely RAPD analysis, anchored inter-SSR PCR, and chloroplast PCR followed by RFLP analysis. The RAPD and inter-SSR markers showed a differentiation between the two subspecies of P. major. These results are discussed in relation to earlier results using allozyme electrophoresis, DNA fingerprinting, and chloroplast RFLP analysis.

Genomic instability in sporadic colorectal cancer quantitated by inter-simple sequence repeat PCR analysis

Genomic instability plays a major role in cancer by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been developed to provide a rapid and reproducible technique for quantitation of the major type of genomic instability observed in sporadic tumors, namely, that manifesting itself as amplifications, deletions, translocations, and insertions. Evaluation of 59 sporadic colorectal cancers by inter-SSR PCR has demonstrated a wide range of instability, independent of tumor stage at diagnosis. Comparison of these data and the results of microsatellite PCR analysis reveals an association of high genomic instability with loss of heterozygosity but no association with the replication error phenomenon arising from defects in mismatch repair.

Comparative analysis of genetic diversity in pea assessed by RFLP- and PCR-based methods

DNA-based molecular-marker techniques have been proven powerful in genetic diversity estimations. Among them, RFLP was the first and is still the most commonly used in the estimation of genetic diversity of eukaryotic species. The recently developed PCR-based multiple-loci marker techniques, which include RAPD, AFLP, Microsatellite-AFLP and inter-SSR PCR, are playing increasingly important roles in this type of research. Despite the wide application of these techniques, no direct comparison of these methods in the estimation of genetic diversity has been carried out. Here we report a direct comparison of DNA-based RFLP with various PCR-based techniques regarding their informativeness and applicability for genetic diversity analysis. Among ten pea genotypes studied, all the PCR-based methods were much more informative than cDNA-RFLP. Genetic diversity trees were derived from each marker technique, and compared using Mantel's test. By this criterion, all trees derived from the various molecular marker techniques, except for the tree derived from inter-SSR PCR, were significantly correlated, suggesting that these PCR-based techniques could replace RFLP in the estimation of genetic diversity. On the basis of this result, AFLP analysis was applied to assess the genetic diversity of a sample of accessions representing the various species and subspecies within the genus Pisum.