Sewing just got scientific. Buckle up.
Gene SOE is a laboratory technique used to ‘stitch’ two pieces of DNA together; why is it spelt ‘SOE’ I hear you cry? That can be your homework. It’s a mystery to me. What I do know is that this technique has been around since the 80’s and is useful for deleting/fusing two DNA fragments together to produce truncated (that means ‘shortened’) proteins and/or two proteins fused together. That’s all very nice, but how does it work?
The Basic Theory
If you want a full grounding in PCR you should read this article we posted previously (Here’s one we made earlier!).
Gene SOE PCR works, firstly, on the principles of PCR; the Polymerase Chain Reaction. PCR is done in a Thermocycler (a PCR machine), which is basically the kettle of your scientific kitchen. Don’t ask me what the spatula is. PCR is a vital part of any lab; in summary it is the in vitro amplification (production) of a targeted DNA segment using Primers (small oligonucleotides) that are complimentary to the ends of the desired DNA molecule. Temperature cycles help unzip (‘melt’) the DNA which allows for the Primers to bind to their specific sites, signalling for the thermophilic DNA Polymerases to do their work. A cooler cycle then lets the single DNA strands zip back up, completing the cycle. See Figure 1 for a more colourful depiction.
- Buffer- To create the correct conditions for the Polymerase enzyme to work,
- dNTP’s- deoxynucleoside Triphosphates. The building blocks of DNA, the bases A, T, G and C,
- MgSO4– Magnesium Sulphate is required for the Polymerase to operate,
- Enzyme- Taq, KOD, Q5; all are thermophilic DNA Polymerases originally discovered in certain species of bacteria that live in high-temperature environments (such as near underwater vents),
- The DNA Template- Acts as a blueprint for the specific production of the desired DNA fragment/gene. Normally, this is an organism’s genomic DNA but it can be any piece of DNA really.
- DMSO- This chemical isn’t required for a successful PCR but it reduces the chances of the Primers ‘tangling up’ and creating secondary structures that can interfere with the process. Many scientists add this as routine; a belts and braces approach.
Principles Of Gene SOE PCR
Building upon this basic technique, a Gene SOE PCR is essentially three separate PCR’s using Primers that are specially designed to have complementary sequences to each other. That is, the Primers for the 1st DNA fragment have complimentary ‘dangling ends’ to the Primers for the 2nd DNA fragment. These dangling ends do not contribute to the complementary binding of the Primer to the Template strand.
Firstly, a PCR is performed on each desired DNA fragment (PCR’s 1 and 2). The products of these reactions, once cleaned and purified, are then mixed and the PCR is run again. This 3rd and final PCR will produce ‘normal’ products at first, however after a few rounds of amplification there will be a splicing of the two unrelated DNA fragments together into one amplified product via the Primer’s complementary sequences. The aim is to get an excess of this fused DNA product so that it vastly outnumbers the unfused fragments until they practically do not factor into the process. For a better idea of this, see Figure 2.
And that is, in a nutshell, it! Gene SOE PCR, everybody. The scientist’s sewing; a quick, reliable method for genetic manipulation in the lab. But hopefully not in the home.
Horton RM, Hunt HD, Ho SN, Pullen JK, & Pease LR (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 77 (1), 61-8 PMID: 2744488
All Figures made by R. Sweet.
Featured Image taken from New Wine Church website http://www.newwinechurch.com/about/our-dna/ accessed at 9.46pm on 18.2.14