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PubMed1993

Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine.

Deficient hydroxylation of debrisoquine is an autosomal recessive trait that affects approximately 7% of the Caucasian population. These individuals (poor metabolizers) carry deficient CYP2D6 gene variants and have an impaired metabolism of severely commonly used drugs. The opposite phenomenon also exists, and certain individuals metabolize the drugs very rapidly, resulting in subtherapeutic plasma concentrations at normal doses. In the present study, we have investigated the molecular genetic basis for ultrarapid metabolism of debrisoquine. Restriction fragment length polymorphism analysis of the CYP2D locus in two families with very rapid metabolism of debrisoquine [metabolic ratio (MR) for debrisoquine = 0.01-0.1] revealed the variant CYP2D6 gene CYP2D6L. Eco RI RFLP and Xba I pulsed-field gel electrophoresis analyses showed that this gene had been amplified 12-fold in three members (father and his two children) of one of the families, and two copies were present among members of the other family. The CYP2D6L gene had an open reading frame and carried two mutations causing amino acid substitutions: one in exon 6, yielding an Arg-296-->Cys exchange and one in exon 9 causing Ser-486-->Thr. The MR of subjects carrying one copy of the CYP2D6L gene did not significantly differ from that of those with the wild-type gene, indicating that the structural alterations were not of importance of the catalytic properties of the gene product. Examination of the MR among subjects carrying wild-type CYP2D6, CYP2D6L, or deficient alleles revealed a relationship between the number of active genes and MR. The data show the principle of inherited amplification of an active gene. Furthermore, the finding of a specific haplotype with two or more active CYP2D6 genes allows genotyping for ultrarapid drug metabolizers. This genotyping could be of predictive value for individualized and more efficient drug therapy.

Alleles↗

PubMed1997

Transgenic medaka fish bearing the mouse tyrosinase gene: expression and transmission of the transgene following electroporation of the orange-colored variant.

Transgenic fish bearing the mouse tyrosinase gene (mg-Tyrs-J) were produced by transfection into fertilized eggs of the homozygous normal orange-colored variant of medaka fish, Oryzias latipes, by means of electroporation. Of 589 eggs transfected, 38 fish (6%) exhibited brownish wild-type skin pigmentation, which was discernible from control siblings. Light microscopy of the skin from the founders thus generated disclosed that 1) melanization occurred and was restricted to melanophores formed presumably from pre-existing amelanotic melanophores, 2) there was a wide variation in the degree of melanization observed among melanophores, and 3) no melanin deposition was recognized in xanthophores or leucophores. Immunofluorescence using an antibody raised against mouse tyrosinase disclosed that melanophores at varying stages of maturation were reactive. Thus, it was shown that the transgene in medaka fish expressed its action in a cell type-specific manner. Crossing of transgenic founders with homozygous orange-colored variant fish yielded two groups of offspring expressing either the wild-type or the orange-colored skin pigmentation at an approximate ratio of 1:1. Crossing between founders exhibiting wild-type pigmentation yielded only offspring with melanized skin. Skin melanophores in these offspring formed vertical stripes, which are rare in this species. The hereditary basis of melanized skin was demonstrated in matings of F1 progenies, which resulted in similar degrees of melanization over whole skin melanophores. The sum of these findings implied that the transgene is expressed as a dominant character gene and is transmitted through germ cell lines according to the Mendelian law. PCR analysis combined with nested PCR technique strongly suggested that the transgene was integrated into the medaka genome, even though the copy number deduced from gel banding was largely diminished, possibly as a result of fragmentation or instability within the medaka genome.

Animals↗

PubMed1990

Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design.

We aligned published sequences for the U3 region of 35 type C mammalian retroviruses. The alignment reveals that certain sequence motifs within the U3 region are strikingly conserved. A number of these motifs correspond to previously identified sites. In particular, we found that the enhancer region of most of the viruses examined contains a binding site for leukemia virus factor b, a viral corelike element, the consensus motif for nuclear factor 1, and the glucocorticoid response element. Most viruses containing more than one copy of enhancer sequences include these binding sites in both copies of the repeat. We consider this set of binding sites to constitute a framework for the enhancers of this set of viruses. Other highly conserved motifs in the U3 region include the retrovirus inverted repeat sequence, a negative regulatory element, and the CCAAT and TATA boxes. In addition, we identified two novel motifs in the promoter region that were exceptionally highly conserved but have not been previously described.

Animals↗

PubMed2005

Prospects of manipulating diatom silica nanostructure.

A key to the development of nanotechnology will be the ability to make complex nanoscaled three-dimensional structures at low cost and in large numbers. The wide variety of structures in the silicified cell walls of diatoms offers a promising natural source of such materials. Diatom silica can be converted into other materials, with maintenance of detailed morphology. To facilitate the use of diatoms in nanotechnology, specific manipulation of the structure in vivo will be desirable. This article explores the possibilities of manipulating diatom silica structure, by nongenetic and genetic means. Nongenetic influences that affect silica structure include changes in environmental conditions and life cycle stages and the presence or absence of particular compounds. The genetically based natural variation in structure in different diatom species indicates that genetic manipulation is possible. To achieve this, however, several goals must be met. The first is to identify cell wall synthesis (CWS) genes involved in structure formation. The recently completed genome sequence of Thalassiosira pseudonana opens the door for genomic and proteomic approaches to accomplish this. An important method to determine the function of CWS genes will be to modify gene sequences or expression and monitor the effect on structure. Performing gene modifications is straightforward, and modified genes can be introduced into diatoms, but the current inability to replace native diatom genes with modified copies could be a problem. However, there are feasible approaches yet to be applied to achieve this goal. It is very likely that continued development and application of molecular genetic techniques will enable us to specifically modify diatom silicified structures and provide a detailed understanding of the underlying mechanism of their formation.

Biomimetics↗

PubMed2000

High copy display of large proteins on phage for functional selections.

We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.

Amino Acid Sequence↗

PubMed2005

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope.

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.

Antibodies, Monoclonal↗

PubMed2005

Markov Chain modeling of pyelonephritis-associated pili expression in uropathogenic Escherichia coli.

Pyelonephritis-associated pili (Pap) expression in uropathogenic Escherichia coli is regulated by a complex phase variation mechanism involving the competition between leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). Population dynamics of pap gene expression has been studied extensively and the detailed molecular mechanism has been largely elucidated, providing sufficient information for mathematical modeling. Although the Gillespie algorithm is suited for modeling of stochastic systems such as the pap operon, it becomes computationally expensive when detailed molecular steps are explicitly modeled in a population. Here we developed a Markov Chain model to simplify the computation. Our model is analytically derived from the molecular mechanism. The model presented here is able to reproduce results presented using the Gillespie method, but since the regulatory information is incorporated before simulation, our model runs more efficiently and allows investigation of additional regulatory features. The model predictions are consistent with experimental data obtained in this work and in the literature. The results show that pap expression in uropathogenic E. coli is initial-state-dependent, as previously reported. However, without environment stimuli, the pap-expressing fraction in a population will reach an equilibrium level after approximately 50-100 generations. The transient time before reaching equilibrium is determined by PapI stability and Lrp and Dam copy numbers per cell. This work demonstrates that the Markov Chain model captures the essence of the complex molecular mechanism and greatly simplifies the computation.

Algorithms↗

PubMed2005

Inferences of evolutionary relationships from a population survey of LTR-retrotransposons and telomeric-associated sequences in the Saccharomyces sensu stricto complex.

The Saccharomyces sensu stricto complex consists of six closely related species and one natural hybrid. Intra- and inter- species variability in repetitive elements can help elucidate the population structure and evolution of these close relatives. The chromosome positions of several telomeric associated sequences (TASs) and LTR-retrotransposons have been determined, using PFGE, in 112 isolates. Most of the repetitive elements studied are found in multiple copies in each strain, although in some subpopulations these elements are present in low copy number or are absent. Hybridization patterns and copy numbers of the repetitive elements correlate with geographic distribution. These patterns may yield interesting clues as to the origins and evolution of some TASs and retrotransposons, e.g. we can infer that Y' originated on the left end of chromosome XIV. There is strong evidence for horizontal transfer of Ty2 between S. cerevisiae and S. mikatae. Ty1 and Ty5 are either lost easily or frequently horizontally transferred. We have also found some gross chromosomal rearrangements in isolates within species and a few new natural hybrids between species, indicating that these processes occur in the wild and are not limited to conditions of human influence. DNA sequences have been deposited with the EMBL/GenBank database under Accession Nos AJ632279-AJ632293.

Base Sequence↗

PubMed2005

Molecular evidence for the existence of natural hybrids in the genus Zygosaccharomyces.

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.

Carboxy-Lyases↗

PubMed1998

Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis.

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.

DNA Restriction Enzymes↗

PubMed2026

Targeted long-read genomic and epigenomic profiling enhances timely comprehensive variant discovery in hypotonia and muscle weakness.

BACKGROUND: Identifying the genetic basis of hypotonia and muscle weakness is critical for patient management and family counseling. However, diagnosis is often hindered by diverse genomic alterations, including repeat expansions, structural variants (SVs), and methylation defects. Standard-of-care testing, largely based on short-read sequencing, is limited in its ability to detect this heterogeneous variation landscape, leaving many patients undiagnosed or requiring lengthy sequential testing. Long-read sequencing represents a promising solution. However, its application as a first-tier diagnostic assay for hypotonia remains unexplored. METHODS: We retrospectively analyzed 227 patients with hypotonia to assess diagnostic yield, time-to-diagnosis, and costs associated with standard-of-care testing. A long-read whole-genome sequencing (LR-WGS) workflow with targeted analysis of hypotonia-associated genes was developed to detect and prioritize pathogenic SNVs, SVs, and CNVs, repeat expansions, and methylation changes at key disease loci. The workflow was validated in a reference-positive cohort with known diagnoses (n = 15) and applied to an unsolved cohort (n = 14). Variant interpretation followed ACMG guidelines and was confirmed with orthogonal methods. RESULTS: Standard-of-care testing achieved a diagnostic yield of 42% with an average time-to-diagnosis of 68.7 days; however, 30% of diagnosed patients experienced significant delays (average 169 days) due to sequential testing. The LR-WGS based approach identified all known pathogenic variants in the positive cohort, including SMN1 deletions, methylation defects at 15q11.2/Prader-Willi locus, FMR1 repeat expansions, and sequence and copy-number variants in > 100 genes underlying myopathies and muscular dystrophies. The targeted long-read pipeline reduced prioritized variant calls by 97.9-99.9% and, in the unsolved cohort, yielded one definitive diagnosis (de novo COL6A3 deletion) and one possible diagnosis (aberrant methylation and copy number at POMK), for an additional 14% yield. Among patients diagnosed after sequential testing (n = 29), LR-WGS is expected to reduce time-to-diagnosis by ~ 85% and decrease cumulative diagnostic delays, with projected healthcare cost savings of $396,000-439,000. Across the entire 227 patient cohort, LR-WGS is anticipated to reduce testing costs by 6.5%, yielding an average savings of $105 per patient. CONCLUSIONS: LR-WGS enables comprehensive discovery of genomic and epigenomic variants in hypotonia and muscle weakness, improving diagnostic yield, shortening diagnostic timelines, and reducing costs compared with current standard-of-care testing.

Humans↗

PubMed1997

Evidence for association between the class I subset of the insulin gene minisatellite (IDDM2 locus) and IDDM in the Japanese population.

Although the shortest (class I) minisatellite (i.e., variable number of tandem repeats [VNTR]) alleles in the 5' region of the insulin gene are positively associated with IDDM in Caucasians, the majority of Japanese are homozygous for class I alleles. Here, we determined the exact length, in number of repeat units (RUs), of class I alleles in Japanese subjects. The distribution of class I alleles in Japanese was trimodal, with peaks located at 32/33, 41, and 44 RUs. The shortest component (i.e., 1S [25-38 RUs]) alleles were significantly increased in the IDDM group compared with the control group (54 vs. 46%; P = 0.040). The 1S/1S genotype was significantly increased in the IDDM patients (34 vs. 20%; P = 0.005; relative risk 2.1). Furthermore, the transmission disequilibrium test of Japanese families with 1S/1M or 1S/1L heterozygous parents confirmed the association of 1S alleles; 17 alleles of 1S and 6 alleles of 1M (39-41 RUs) or 1L (42-44 RUs) were transmitted to affected offspring (P = 0.022). In addition, we found tight linkage of 1S with allele 9 of the tyrosine hydroxylase gene microsatellite and allele (-) of the IGF-II gene Apa I polymorphism, but neither 9 nor (-) alleles were significantly associated with IDDM. The present study suggests that a class I subset may have a role in IDDM susceptibility in Japan. It was revealed that the difference between 1S alleles and 1M or 1L alleles is almost consistently characterized by a sequence variation generated by deletion of two copies of an ACAGGGGTCC CGGGG repeat element, implying that sequence variation of class I alleles may influence disease susceptibility.

Adolescent↗

PubMed1996

The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers.

The giant sarcomeric protein titin (also described as connectin) is composed mainly of immunoglobulin (Ig)-like and fibronectin type III (fn3)-like domains arranged consecutively. At both ends of the molecule, these domains are interrupted by sequence insertions. The amino terminus of titin is localized in the Z-disk, a structure of great variability in different muscle types. We have determined the ultrastructural position of sequences in this region of the molecule in skeletal and cardiac muscle by immunoelectron microscopy using antibodies directed against unique epitopes. Titin molecules entering the Z-disk from two half sarcomeres do not significantly overlap, showing that the amino terminus is at the centre of the Z-disk. A serine/proline rich site, which can be phosphorylated by kinases in developing muscle tissues, was identified near the amino terminus of titin. Sequence analysis revealed the presence of a novel 45 residue repeat ('Z-repeats') in this region of the molecule. The number of titin Z-repeats varies due to differential splicing. We propose that this mechanism is a means of assembling Z-disks of variable thickness and mechanical strength.

Amino Acid Sequence↗

PubMed1976

Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation.

Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, approximately 4.4 X 10(6), approximately 20 copies per chromosome; RHO = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 TO B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill + transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPl10- determined killing activity. Plasmid pPl10 was therefore inserted into B-subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoR1.

Bacillus↗

PubMed1990

A retroposon in the 5' flank of a Trypanosoma brucei VSG gene lacks insertional terminal repeats.

A retroposon-like repeated sequence, ingi, occurs in high copy number in the genome of Trypanosoma brucei brucei. An ingi is present in the 5' flank of the 5C gene, an intrachromosomal IsTat 1.5 variant surface glycoprotein (VSG) gene family member. The 5' end of the ingi is located 22 bp upstream of the putative VSG start codon and the ingi open reading frame is in the opposite orientation to that of the VSG gene. The termini of the ingi are not flanked by a short repeat sequence and there are no sequences upstream of the ingi insertion which are homologous to the 5' flanking sequence of other 5 VSG gene family members. Thus, it appears that recombination and/or gene conversion between two ingi sequences may have eliminated the original 5C gene flanking sequence. Similar events may also have occurred with all but one previously reported ingi.

Amino Acid Sequence↗

PubMed1997

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Amino Acid Sequence↗

PubMed1999

Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes.

A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before.

Animals↗

PubMed2001

[Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR)].

Due to its extraordinarily high sensitivity, polymerase chain reaction (PCR) is widely used for amplifying cDNA copies of low abundance mRNA. However, quantitation is unreliable because the amount of PCR product increases exponentially with each cycle of amplification; therefore, minute differences in any of the variables that affect the efficiency of amplification can dramatically alter product yield. Coamplification with a different reporter gene product is a semiquantitative mRNA quantitation method. Thus, we constructed an internal standard with a defined deletion fragment from the target cDNA, and used the same primers to coamplify the unknown and the competitor, allowing us to quantify the amount of specific target cDNA available. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube-to-tube or sample-to-sample variations in the kinetics of the RT reaction. For RNA quantitation, Northern blots are widely used. However, the Northern blot technique requires at least 10mg of total RNA for semiquantitation while QC-PCR requires only 1mg of total RNA and is useful when only small amounts of tissue are available. QC-PCR is a simple, and inexpensive method in which competitive PCR is used for highly accurate quantitation of mRNA and DNA from a small number of cells.

Blotting, Northern↗