实验概要

        Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing.Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product.Internal primers generate overlapping, complementary 3‘ ends on the intermediate segments and introduce nucleotide substitutions,insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3’ region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.

实验原理

        Modification of genes at the nucleotide level continues to provide relevant insights into the structural elements critical to gene and protein function. Site-directed mutagenesis generates targeted changes including single or multiple nucleotide insertions, deletions or substitutions, generally via the use of an oligonucleotide primer that introduces the desired modification. Another targeted approach to elucidate the structural basis of function is the generation of chimeric molecules by splicing together different regions of one or more genes. Here, we outline a PCR-mediated method of extending overlapping gene segments that has proven useful for site-directed mutagenesis, the creation of chimeric molecules or even the cloning of large gene segments by splicing together smaller pieces1–5.This PCR-based technique was developed in our laboratory in the late 1980s (see refs. 6,7) and provides several advantages over site-directed mutagenesis strategies that existed at that time. In contrast to protocols using single-stranded8 or double-stranded DNA plasmids9,10, this strategy enables the target gene to be amplified from genomic DNA or cDNA and does not require cloning the gene of interest into a plasmid before mutagenesis. Instead, for site-directed mutagenesis (Fig. 1a), segments of the target gene are amplified from the template DNA using two flanking master primers (a and d) that mark the 5‘ ends of both strands and two internal primers (b and c) that introduce the mutation of interest (indicated by the cross). Precise design of these primers is critical to the success of this procedure. For example, internal primers must not only contain the desired mutation, but must also create overlapping nucleotide sequences. In this manner,when the strands of products AB and CD are denatured during the second PCR, strands with 3’ complementary ends created by mutagenic primers b and c will anneal. Primers a and d will amplify one of these hybridization products (sense strand from AB and antisense strand from CD) to generate the predominant product AD, whereas the other hybridization product (sense strand CD and antisense strand from AB) will not be amplified in the absence of driving primers. Similarly, although the sense and antisense strands of each product AB and CD will also rehybridize, without primers band c to increase their copy number throughout the PCR cycles,they are not major products of this PCR. In summary, master primers a and d will preferentially amplify product AD with the intended mutation.

主要设备

        Thermocyler PCR machine

        PCR tubes that accommodate at least 50 ml volume and fit into the PCR machine

        Combs and gel tray to pour agarose gels

        Gel electrophoresis apparatus

        Power source to supply gel electrophoresis apparatus

        UV light source
        Spectrophotometer

实验材料

实验步骤

（1）Label PCR tubes for two negative controls and products AB and CD. Combine reagents in individual PCR tubes on ice. Add the Taq polymerase last, keeping it at -20 ℃ until its addition. Negative control reactions do not include template DNA; thus,no PCR product should be amplified in these reactions, unless one or more  of the other reagents (including primers) are contaminated with DNA.

（2） Place tubes in PCR machine and run using the program below:

（3） Pour a 1% (w/v) agarose gel by melting 1 g of agar in 100 ml of 1
TAE buffer in the microwave. When the mixture has cooled (flask should be warm, not hot to touch), add 11 ml saturated aqueous ethidium bromide. Swirl gently to mix, but do not splash. Pour mixture into a gel tray containing a comb with wells that can accommodate at least 60 ml volume. Allow the gel to solidify at room temperature (20–25℃).

（4） Place the solidified gel into the gel electrophoresis apparatus and remove the comb. Cover the gel with 1×TAE buffer and ensure that buffer fills each end of the electrophoresis apparatus.

（5）Load 10 ml DNA ladder into the first well. Add 5 ml of 10
Ficoll to each 50 ml PCR and load the entire amount into the gel. Skip a lane between samples in order  to space out the resulting bands, avoid spillover from one lane to the next and make excision of bands easier.

（6） Run the gel at 80–100 V, periodically monitoring its progress with a UV light source. Continue to run the gel until the DNA ladder has resolved and bands from the PCR samples have separated well enough to confirm the size of the band of interest.Achieving complete resolution of the PCR products is important to prevent contamination during excision of the band of interest.To avoid running the gel for a longer time and potentially losing DNA product, at the latest, the run should be   stopped once the dye front reaches approximately 1 cm from the bottom.

（7）Confirm the expected size of the PCR products by comparing with the DNA ladder, and then carefully excise the bands containing the products with a razor blade and place into separate microfuge tubes. If the weight of a gel fragment exceeds 0.4 g, cut the fragment into smaller pieces and place into additional tubes. It is not necessary to use different razor blades for the extraction of different DNA bands.

（8）Purify the PCR products using either the Gene Clean Purification kit or the QIAquick Gel Extraction kit. In either case,follow the manufacturer’s protocol.

（9）Quantify DNA (eluted in a 30 ml volume) using a spectrophotometer. At least 50 ng DNA is required of each product AB andCD for the next PCR. DNA quality and cleanliness can also be assessed at this time from optical density readings by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm (A260/280) for each sample. An A260/280 ratio ranging between 1.7 and 2.0 is indicative of good quality and decent DNA purity. Note: we have also had success with subsequent PCRs using DNA of lesser quality (A260/280 o1.7).

（10）Calculate the volumes of PCR product templates AB and CD, primers a and d, and water to be used in each PCR. Prepare each PCR by combining the  appropriate reagents in PCR tubes on ice, as tabulated below, again keeping the Taq polymerase cold and adding it last. A negative control reaction without  template is included, as in Step 1, to detect any reagent contamination.    

（11）Place tubes into PCR machine and run the PCR program as outlined in Step 2, adjusting annealing temperature if required.
（12）Repeat Steps 3–9 to purify the final PCR product AD. 

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