Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5′-segment that initiates stable priming, and a short 3′-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.

We developed GeneFishing technology, an improved method for the identification of differentially expressed genes (DEGs) using our novel annealing control primer (ACP) system. Because of high annealing specificity during PCR using the ACP system, the application of the ACP to DEG discovery generates reproducible, authentic, and long (100 bp to 2 kb) PCR products that are detectable on agarose gels. To demonstrate this method for gene expression profiling, Gene-Fishing technology was used to detect genes that are differentially expressed during development using total RNAs isolated from mouse conceptus tissues at 4.5-18.5 days of gestation. Ten DEGs (DEG1-10) were isolated and confirmed by Northern blot hybridization. The sequence analysis of these DEGs showed that DEG6 and DEG10 are unknown genes.

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3 end target core sequence and the 5 end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5 end nontarget sequence to the template and facilitates primer hybridization at the 3 end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.

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To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5-flanking region (؊2100 to ؉67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene. Fatty acid synthase (FAS)1 plays a central role in de novo lipogenesis in mammals and birds by catalyzing all the reactions in conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS activity is not known to be regulated by allosteric effectors or covalent modification. However, FAS concentration in liver and adipose tissue is highly sensitive to nutritional, hormonal, and developmental states (1-3). When rats are fasted for 1-2 days, the rate of synthesis of FAS declines, while refeeding a high carbohydrate diet increases synthesis of FAS (4). We have previously reported that FAS mRNA was not detectable in livers of fasted mice but dramatically increased upon refeeding a high carbohydrate, fat-free diet (5). This induction of FAS mRNA resulted...