Uniform Terminus PCR: Amplification of Minute Unknown DNA Fragments

Most published methods for obtaining unknown DNA fragments involve ligation-mediated polymerase chain reaction (PCR) that allows the amplification of unknown sequences flanking short known fragments of DNA. The methods require using a single specific primer (or a pair of nested primers) and a nonspecific primer complimentary to either plasmid sequence or a linker DNA duplex ligated to blunt or sticky DNA ends (1–5). Here, we report an easily implemented modification for amplifying unknown DNA fragments that is named uniform terminus PCR (UT-PCR). We have successfully used this method to amplify and clone DNA fragments from different genes. As an example, we describe the amplification of the (CTG)n repeats of the 3′-untranslated region (3′-UTR) in human myotonin protein kinase (MT-PK) gene. This method has also been used to analyze the termini of PCR products amplified by using Taq DNA polymerase. The key point of the modification is to synthesize a linker with complete or incomplete palindromic structure as its stem part and three random nucleotides at its 3′ end (PPL in our experiment), and an amplification primer containing only the sequence of the stem part of the linker (PP in our paper). The 5′ region of the PPL has been previously phosphorylated to facilitate its ligation to the single-stranded (ss)DNA to be amplified. The last nucleotide in its 3′ end is a ddNTP, which avoids the linker/linker ligation; and during PCR cycling, the extension from the linker residues left from the ligation step is blocked. The 3′ protruding end of the linker improves ligation efficiency greatly and permits its application to both double-stranded (ds)DNA and ssDNA. The palindromic structure of the linker assures the 3′-NNN end anneals to the 3′ end, rather than other sites of the template DNA, which is important for obtaining full-length PCR products of interest. During the amplification, the palindromic structure in the primer leads to the annealing of most of the primers, leaving limited amount of free primer molecules. The latter can anneal more specifically to the stem part of the linker ligated to the 3′ end of ssDNA, therefore greatly raising the specificity of UT-PCR. The three restriction sites in the primer facilitate the cloning after amplification. The PPL molecules are incubated at 65°C for 10 min and slowly cooled down to room temperature to form an almost perfect double-stranded structure, leaving the three random nucleotides free. Figure 1 shows the annealing reaction. It is the 3′-NNN in the annealed double-stranded PPL that can anneal to any ssDNA, irrespective of the DNA fragments being blunt or sticky, and their sequence being known or not, which makes the subsequent amplification possible and effective. Thus, only one primer has to be made, and this primer is used for any UT-PCR application. First, the DNA fragments to be amplified were heat-denatured to generate ssDNA. Then, the dsPPL was annealed and ligated to the denatured ssDNA. Third, the ligated DNAs were denatured in the presence of PP. Finally, the PP served as an amplification primer, and the PCR was carried out. Briefly, about 30 ng (ca. 0.3–0.5 pmol) of heatdenatured PCR products of (CTG)n repeats, with flanking sequences in MTPK gene amplified from genomic DNA, were mixed with 2 U of T4 DNA ligase in a 20–30-μL reaction volume containing 1× Ligation Buffer (Life