Combination Primer Of Arbitrary And Amchored Primer

Science (1996) 274, 610-614

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The messenger RNAs (mRNAs) within the total RNA population are used as the templates for DD-PCR after first-strand cDNA synthesis by reverse transcription. The current methodology makes use of three "anchored" oligo-dT primers that target the poly-adenylation site of eukaryotic mRNA and have the form H-T11M, where H is a HindIII restriction site (AAGCTT), T11 is a string of 11 Ts (though the first two Ts come from the HindIII site), and M is G, C, or A (9). They are referred to as "anchor" primers because the non-T base after the string of 11 Ts enables the primer to be anchored to the same spot for each round of amplification, in contrast to standard oligo-dT primers that only contain a string of Ts and will anneal in multiple spots, creating a smear (see Note 1). The HindIII restriction site was added to the anchor primer design to make the primers longer and more efficient in annealing to the targeted poly-A site, as well as improving downstream applications such as cDNA cloning. Using the current anchor primer design, the cDNA populations are subsequently divided into three subpopulations that represent one-third of the potential mRNA expressed in the cell at any given time. Previous work indicated using anchor primers of the type T11VN, where V can be A, G, or C and N can be any of the four nucleotides, as well as anchors of the type T12MN, where M is a degenerate mixture of A, G or C, and N is any of the four nucleotides (1). Both of those primer designs result in larger subfractions of the mRNA population (12 for type T11VN and 4 for T12MN), which unnecessarily increases the amount of FDD-PCR reactions for the same level of gene coverage vs the H-T11M primer design.

The next step in DD is the PCR-amplification of the cDNA subpopulations utilizing a combination of anchor primers (called H-T11M) with a set of "arbitrary" primers that are random and short in length. The design of these arbitrary 13mers (H-AP primers) utilized in DD technology also includes a HindIII restriction site (AAGCTT) and a 7-basepair backbone of random base combinations. The HindIII restriction site is included in both the anchor and arbitrary primers for more efficient primer annealing and easier downstream manipulation of the cDNA (9). The primers used in DD represent a random selection from over 16,000 (47) basepair combinations. Additionally, the length of an arbitrary primer is so designed that by probability each will recognize 50-100 mRNAs under a given PCR condition (10). As a result, mRNA 3' termini defined by any given pair of anchored-primer and arbitrary primer are amplified and displayed by denaturing polyacrylamide gel electrophoresis (PAGE). A mathematical model of estimated gene coverage utilizing various combinations of anchor and arbitrary primers was developed shortly after the advent of DD technology (10). This mathematical model indicated that approx 240 primer combinations (three anchor primers with 80 arbitrary primers) were needed to approach the level of estimated genome-wide screening for eukaryotes (approx 95%). However, a new mathematical model presented in the previous chapter of this book, predicts that more primer combinations are required to give that level of coverage; using 480 primer combinations (3 anchor primers with 160 arbitrary primers) would provide approx 93% coverage based on the new model.

DD was originally optimized with radioactivity using 35S (1). 33P labeling was then developed (9) for better sensitivity and resolution and has been the most commonly used for publications. However, fluorescent differential display (FDD) (see Fig. 1) was the next logical progression. In the development of FDD, it was crucial that the new platform have similar sensitivity to traditional DD with isotopic labeling, as well as other advantages that would make the platform a viable and improved alternative to the established DD methodology. FDD, optimized using fluorochrome-labeled anchor primers (generi-cally called FH-T11M) and higher dNTP concentrations in PCR, was shown to be essentially identical in both sensitivity and reproducibility to that of conventional DD (6) (see Fig. 2). Improvements such as elimination of radioactivity, digital data acquisition, and increased assay speed were goals that were successfully reached by the establishment of the FDD platform, representing a marked improvement over conventional DD.

After PCR amplification, gel electrophoresis is performed to separate the resulting PCR products by size. Reactions are run side by side so that the samples being compared are next to one another for each primer combination. Comparison of the cDNA patterns between or among relevant RNA samples reveals differences in the gene expression profile for each sample (see Fig. 3). Electrophoresis can be performed with denaturing polyacrylamide sequencing gels (1,11), nondenaturing polyacrylamide gels (7), or with agarose gels (12,13). Sequencing gels are the most commonly used method and are recommended here because they offer the best band resolution and allow for easy and efficient recovery of genes. In addition, their ability to accommodate a large number of reactions reduces the number of gels that must be run for FDD analysis.

Fig. 1. Schematic representation of fluorescent mRNA differential display. Three fluorescently labeled one-base anchored oligo-dT primers with 5' HindIII sites are used in combination with a series of arbitrary 13mers (also containing 5' HindIII sites) to reverse transcribe and amplify the mRNAs from a cell.

Fig. 1. Schematic representation of fluorescent mRNA differential display. Three fluorescently labeled one-base anchored oligo-dT primers with 5' HindIII sites are used in combination with a series of arbitrary 13mers (also containing 5' HindIII sites) to reverse transcribe and amplify the mRNAs from a cell.

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