Identification of the gene-richest bands in human chromosomes

Saccone, Salvatore; Cacciò, Simone M.; Kusuda, Jun; Andreozzi, L.; Bernardi, Giorgio

doi:10.1016/0378-1119(96)00392-7

Cited by 110 publications

(99 citation statements)

References 36 publications

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“…19,20 This high GC content is partly attributable to transposable element insertions and regional gene density. 19,21,22 It has been noted that the GC content of DNA can significantly affect polymerase chain reaction amplification efficiency, sometimes causing premature chain termination at the beginning of G(C)-rich regions. [23][24][25][26] Although the exact mechanism of how GC content may affect DNA polymerase function is not well understood, it has been hypothesized that amplification of GC-rich templates can be hampered because of the formation of secondary structures such as hairpins and by higher melting temperatures, which can inhibit primer extension by DNA polymerases as well as enzyme processivity.…”

Section: Discussionmentioning

confidence: 99%

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Bredel

Juric

et al. 2005

The Journal of Molecular Diagnostics

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Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffinembedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalinfixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fineneedle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. The availability of a simple and reliable method for the amplification of entire genomes from limited sources of DNA would lend itself to high-throughput genetic analyses in virtually all areas of translational research. High-throughput genomic profiling, for example cDNA microarray-based comparative genomic hybridization (array-CGH), 1 requires g quantities of genomic DNA. A particular challenge for translation of array-CGH methodology to clinical application is to link it to a robust up-front technology that allows reliable and unbiased amplification of limited sources of DNA, for example from fine-needle biopsies. Much effort has been invested in developing methods for whole genome amplification, for example polymerase chain reaction-based methods. [2][3][4] In particular, the pitfalls of substantial variation in the extent of amplification occurring between different markers, incomplete representation, and inadequate average DNA size have limited the use of most existing amplification methods, making them particularly unsuitable for genomic applications. 2-4Bacteriophage Phi29 DNA polymerase random-primed DNA amplification 5-7 is based on an isothermal strand displacement amplification reaction in which random hexamer primers anneal to the genomic template at multiple sites and Phi29 initiates replication at these sites on the denatured linear DNA. As synthesis proceeds, strand displacement of complementary DNA generates new single-stranded DNA available to be primed by additional primers. The subsequent strand displacement replication of this DNA leads to the formation of double-stranded DNA. The presence of an associated proofreading activity of Phi29 ensures a high sequence accuracy of the amplified DNA, indicating its suitability f...

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Section: Discussionmentioning

confidence: 99%

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Bredel

Juric

et al. 2005

The Journal of Molecular Diagnostics

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“…Our results also indicate that the high genetic instability applies to the other loci in the 1p region; indeed, 26 of 28 HCC cases (83%) had an alteration in at least one locus within the 1p22-pter region. A recent study suggests that 1p36 is a genomic region with high gene density, 44 §M:F ratio was calculated from the number in each category.…”

Section: Discussionmentioning

confidence: 99%

Multiple genetic alterations, 4q28, a new suppressor region, and potential gender differences in human hepatocellular carcinoma

Hammond¹,

Jeffers²,

Carr³

et al. 1999

Hepatology

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Primary hepatocellular carcinomas (HCCs) of different etiologies were studied to determine the rate of alteration of several genetic regions previously associated with the HCC phenotype. The focus of our study was to identify the frequency of genetic alterations within individual HCCs and their distribution among male and female cases. Genetic differences were evaluated between DNA isolated from tumor (T) and corresponding non-tumor (N) tissue using short tandem repeat (STR)-microsatellites and restriction fragment length polymorphism (RFLP) analyses. Twenty-eight HCC cases were studied with polymorphic markers from different parts of the genome. Three or more loci were identified with genetic alterations from 28 loci tested in 63% of HCC cases. Human hepatocellular carcinoma (HCC) remains a leading cause of cancer death worldwide. HCC has a very poor prognosis, and there is only limited understanding of the molecular events that lead to uncontrolled proliferation of the liver parenchymal cells. Several etiologic factors have been identified as high risk factors in association with HCC development, including previous infection with hepatitis B virus (HBV), hepatitis C virus (HCV), exposure to aflatoxin, and alcohol-induced cirrhosis. 1-4 However, it remains unclear how these agents affect hepatocyte growth, the transformation process, or both.Current research has focused on identifying the genetic region(s) in which a mutation leads to transformation in the hepatocyte and malignant proliferation in the liver because neoplasia is thought to result from the initiation and accumulation of genetic mutations in a multistep process. Genetic evolution of the tumor phenotype is best defined in colorectal cancer in which several sequential mutations have been identified in association with the stage of the disease. 5,6 Alterations of several chromosomal regions have been identified in HCC. 7-38 Some data suggest that certain HCC causes are associated with specific genetic alterations. 25,26,36 p53 is the only gene in which specific mutations have been associated with HCC, but even these mutations are recognized as later alterations in HCC development. [26][27][28][29] Epidemiological studies document the higher incidence of HCC among men than women but so far no correlation with genetic data was implicated. A recent study in 120 HCCs documented frequent loss of heterozygosity (LOH) in chromosome regions 1p, 1q, 2q, 4q, 6q, 8p, 9q, 16p, 16q, and 17p. 34 Despite a long list of loci with frequent LOH in HCCs the sequence of these mutations in HCC development is not understood.We and others have identified a high incidence of genetic alteration in the short arm of chromosome 1 in HCCs of different causes. [30][31][32][33] We now report that the majority of our HCC cases have multiple genetic alterations. The most frequent target for genetic change in HCCs analyzed is the 1p36 region. The frequent LOH in 1p36.1, 1p36.3, 4q28, 13q14, and 17p13 indicates important HCC suppressors in the vicinity of these regions. The 4q28 locus...

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“…In fact, 8 of the 14 orphan contigs contain at least one gene-or EST-specific STS. These findings suggest that many (or most) of the orphan contigs likely correspond to gene-rich regions of the chromosome that are GC-rich, which have been shown to be difficult to clone in YACs (Bernardi 1995;Saccone et al 1996). Supporting this theory is the fact that the average GC content of the STSs in the anchored contigs is 40%, whereas the average GC content of the STSs in the unanchored contigs and those failing to identify a positive YAC is 47%.…”

Section: Unanchored Contigsmentioning

confidence: 91%

A Physical Map of Human Chromosome 7: An Integrated YAC Contig Map with Average STS Spacing of 79 kb

Bouffard¹,

Idol²,

Braden³

et al. 1997

Genome Res.

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The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a ∼170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers ordered by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are ''anchored'' on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain >98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for ∼1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 ''orphan'' contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in >95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of ∼79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

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Identification of the gene-richest bands in human chromosomes

Cited by 110 publications

References 36 publications

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Multiple genetic alterations, 4q28, a new suppressor region, and potential gender differences in human hepatocellular carcinoma

A Physical Map of Human Chromosome 7: An Integrated YAC Contig Map with Average STS Spacing of 79 kb

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