GC content

Summary  Random amplified polymorphic DNA markers were used to distinguish and characterize 20 Indian potato cultivars. A total of 198 scorable fragments were amplified using 10 random primers, only two of which were monomorphic. Similarity values among the cultivars ranged from 0.33 to 0.80. A primer having resolving power above 7.4 was sufficient to distinguish all 20 cultivars. Wide variations in

Many techniques in molecular biology depend on the oligonucleotide melting temperature (T(m)), and several formulas have been developed to estimate T(m). Nearest-neighbor (N-N) models provide the highest accuracy for T(m) prediction, but it is not clear how to adjust these models for the effects of reagents commonly used in PCR, such as Mg(2+), deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO). The experimental T(m)s of 475 matched or mismatched target/probe duplexes were obtained in our laboratories or were compiled from the literature based on studies using the same real-time PCR platform. This data set was used to evaluate the contributions of [Mg(2+)], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical formulas based on GC content, length, and the equivalent sodium ion concentration of cations [Na(+)(eq)] were obtained by multiple regression. When we used [Na(+)(eq)] = [Monovalent cations] + 120(square root of ([Mg...

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (∼32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs2SO4-Ag+ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C0t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a “short period” interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.

Background Transcriptome sequencing (RNA-Seq) has become the assay of choice for high-throughput studies of gene expression. However, as is the case with microarrays, major technology-related artifacts and biases affect the resulting expression measures. Normalization is therefore essential to ensure accurate inference of expression levels and subsequent analyses thereof. Results We focus on biases related to GC-content and demonstrate the existence of strong sample-specific GC-content effects on RNA-Seq read counts, which can substantially bias differential expression analysis. We propose three simple within-lane gene-level GC-content normalization approaches and assess their performance on two different RNA-Seq datasets, involving different species and experimental designs. Our methods are compared to state-of-the-art normalization procedures in terms of bias and mean squared error for expression fold-change estimation and in terms of Type I error and p-value distributions for tes...