ProductInformation INTRODUCTION TO THE VECTORETTE SYSTEM

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1 INTRODUCTION TO THE VECTORETTE SYSTEM ProductInformation The following is background information on the Vectorette System, included to familiarize the researcher with the Vectorette Unit and its function within the Human Genomic Libraries. The polymerase chain reaction (PCR) has revolutionized molecular biology (Saiki et al. 1988). The ability to amplify specific fragments of DNA is used in a wide range of applications in all aspects of life science research. Typically, PCR requires sequence knowledge of the regions flanking the gene of interest in order for amplification to occur. For a large number of applications it would be useful to amplify DNA fragments where the sequence of only one end is known. In the past decade, researchers have been using different strategies known as asymmetric PCR or unidirectional PCR to perform this feat (Garrity et al. 1995). The Vectorette PCR system is based on the principle of unidirectional PCR, and allows specific amplification of any uncharacterized sequence adjacent to a known region (Lilleberg et al. 1998). Vectorette PCR technology was invented and patented in 1988 and has since been used extensively for research and development in the genomics field. Since the introduction of the Vectorette I and II systems, Sigma-Genosys has received a great deal of positive feedback and suggestions. In this new, updated Vectorette system, Sigma-Genosys has made substantial improvements based on customer s suggestions to provide superior performance and convenience when compared to competitors systems. These improvements include the use of Hot Start, long and accurate PCR reagents, positive controls, extensively tested protocols and helpful technical tips. PRINCIPLE OF THE VECTORETTE SYSTEM Vectorette units are specially designed double stranded DNA fragments that have a stretch of mismatched nucleotides in the middle. A range of Vectorette units are available that are designed with an appropriate overhang to allow ligation with a variety of specific restriction libraries. These various Vectorette units allow flexibility in the choice of PCR products generated from a particular locus. More information about Vectorette can be obtained at our website, under Molecular Biology Kits, Cloning Subsection. _ 1

2 Figure 1: Schematic of the overall Vectorette process The Vectorette system consists of three key steps as seen in Figure 1: 1. Generation of a Vectorette library by digestion of DNA with a restriction enzyme followed by ligation of the appropriate Vectorette units to the restriction digested DNA fragments. This step has already been accomplished for you in the Human Vectorette Library. 2. PCR using one primer directed at the Vectorette unit and a custom primer targeting the known DNA sequence (the initiating primer). This allows PCR amplification of DNA between the known sequence and the restriction site used to cut the target DNA. _ 2

3 3. Agarose gel fractionation of the PCR product and subsequent sequencing of product of the expected length can be accomplished to verify the correct DNA sequence was amplified. _ 3

4 Vectorette works because the Vectorette primer is identical to the bottom strand of the mismatched portion in the Vectorette adaptor. Therefore, the Vectorette PCR primer has no complementary strand to anneal to in the first cycle of PCR and amplification is completely dependent on first strand synthesis. The initiating primer from the known sequence (directed towards the sequence of interest) will produce a strand complementary to the bottom strand of the Vectorette in the first cycle of PCR. In the second cycle of PCR there is now a template for the Vectorette PCR primer. This template contains the initiating primer from the known sequence at one end and the Vectorette sequence at the other end. After the second cycle, PCR continues normally to amplify the targeted sequence, thereby amplifying previously unknown sequence.. The DNA sequences used in all the Vectorette products have been checked against Genbank databases to ensure minimum cross-homology problems for any target DNA sequence to be amplified and thus increasing the specificity of Vectorette PCR primers. FEATURES OF VECTORETTE UNITS The basic design of the Vectorette unit and the location of the PCR and sequencing primers are shown in Figure 2. I. Construction of the Human Vectorette Libraries Each library is constructed by performing a restriction enzyme digest and annealing it to a vectorette library. a. Bgl II Libraries are made by digesting human genomic DNA with Bgl II enzyme, then ligating with the Bam HI vectorette unit (compatible with enzymes leaving a 5 -GATC overhang. b. EcoRI Libraries are made from EcoRI digest ligated to EcoRI vectorette units (compatible with enzymes leaving a 5 -AATT overhang). c. HindIII Libraries are produced using HindIII digested DNA ligated to HindIII vectorette ends (compatible with enzymes leaving a 5-AGCT overhang) d. PvuII Libraries are made from human genomic DNA digested with PvuII and then ligated to Blunt Vectorette ends. One feature of the sequence at the end of the Vectorette is that although it is ligatable to a particular sticky/blunt end, the original restriction site is not reformed in the target-vectorette construct. Therefore, digestion of a product amplified from, for example, an EcoRI digest can be cloned using an EcoRI/blunt vector, since no internal EcoRI site occurs on the amplicon. _ 4

5 Vectorette Eco RI end = In a ligated construct = 5 -AATTG-3 3 C-5 5 -AATTG-3 3 -TTAAC-5 The Eco RI recognition sequence extends outside of the cut site. Since the recognized bases have changed Eco RI will not cut _ 5

6 II. The mismatched region of the Vectorette. The unpaired region of the Vectorette unit is crucial to its function. The Vectorette PCR primer, which is included in the kit, has been synthesized to have an identical sequence to the bottom strand of the mismatched portion of the Vectorette unit. This increases specificity, since the Vectorette PCR primer has no sequence to anneal to unless a complimentary strand has been synthesized using another (i.e. specific) primer. Geometric amplification, which requires the existence of two oppositely directed primers, depends on the first strand synthesis. Therefore, the only PCR products formed are those that contain the initiating primer. Three restriction sites (Bam H1, EcoR I and Hind III) have been incorporated in the bottom strand, which could facilitate cloning of the final PCR product if necessary (see Figure 2). III. The completely paired regions of the Vectorette. These regions are exactly complementary to each other and provide the double-stranded region of the Vectorette unit. The double-stranded regions in the Vectorette stabilize the unit, forming a partially double-stranded molecule in solution. _ 6

7 Figure 2: Makeup of the Vectorette Adaptor 9(&725(77( Top strand Bottom Strand EcoR I BamH Hind III 5HVWULFWLRQÃ6LWHV REFERENCES Garrity, P. A., Ligation-Mediated PCR, in PCR 2 A Practical Approach, McPherson, M. J., et al., (Eds.) pp Oxford University Press, New York (1995) Lilleberg, S. and Patel, S. Isolation of DNA flanking retroviral integration sites using Vectorette II. Genosys Origins, II (1998) Rychlik, W. Selection of primers for polymerase chain reaction, Mol. Biotechnol. 3, , (1995a) Rychlik, W., Priming Efficiency in PCR. Biotechniques, 18, 84 (1995b) Saiki, R. K., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 (4839), (1988) Sambrook, J., et al. Molecular Cloning: A Laboratory Manual, third edition, Cold Spring Harbor Laboratory. Cold Spring Harbor (2000) (Product Code M8265) Don, R.H., et al., Touchdown PCR to circumvent spurious priming during gene amplification. Nucl. Acid. Res., 19, 4008 (1991). _ 7