Chapter 1 The Early Days of Blotting Edwin Southern Abstract The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA fragments to a membrane support through capillary action, is also known as Southern blotting. This procedure enables the detection of a specific DNA sequence by hybridization with probes. The term Southern blotting led to a “geographic” naming tradition, with RNA blotting bearing the name Northern blotting and protein transfer to membranes becoming known as Western blotting. Key words Southern blotting, Electrophoresis, Western blotting, Northern blotting

1

Introduction It is surprising to me that blotting is still taught and even used for more than 40 years after I did the first one in 1973. At the time I was looking for a way of purifying genes from eukaryotic DNA. The story began with the discovery by Peter Ford that the 5S genes of Xenopus laevis are differentially expressed in oocytes and somatic cells: Peter and I sequenced the 5S RNAs and found that their sequences were different [1]. There was a lot of interest at the time in understanding the molecular mechanisms that underpin the control of gene expression, and this seemed to be a case where the genes might be accessible, as they are highly repeated. There were few ways to purify genes in those days—cloning was not available— but the type II restriction endonucleases had recently been described by Tom Kelly and Ham Smith [2]. Combined with agarose gel electrophoresis, these enzymes provided a powerful way of fractionating DNA. The difficult part was finding the bands of 5S genes in the complex smear of fragments that result from running the digest of eukaryotic DNA on a gel. Cutting the gel into slices, eluting the DNA, and analyzing the fractions by filter hybridization were tedious and gave very noisy backgrounds that hid the low signal from the genes. With Julia Thompson, we tried drying the gel to

Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_1, © Springer Science+Business Media New York 2015

1

2

Edwin Southern

trap the fragments, and hybridizing the dry film with radioactive 5S RNA; this did not work in our hands; and though Oliver Smithies and his colleagues succeeded [3], this method does not seem to have taken off. We shelved the project after Julia left for Canada, but I returned to it when I heard Charlie Thomas describe his way of dissolving agarose gels in concentrated sodium perchlorate [4]. It occurred to me that if we could dissolve the gel while it was laid against a filter, the DNA would stick to the filter, retaining the pattern of bands. The filter-bound DNA could then be hybridized using the well-known Gillespie and Spiegelman method [5]. So I set up a test with a strip of gel floating on a raft of cellulose nitrate filter on a solution of sodium perchlorate. As I sat and watched, I did not see any sign of the gel dissolving, but I did notice a bead of liquid forming on top of the gel. This was the light-bulb moment. I realized that the gel was permeable and that it should be possible to soak out the DNA by a blotting process; it was a matter of putting the filter on top of the gel, with an overlay of filter papers, and substituting SSC for sodium perchlorate. This worked first time and it was a thrill to see the sharp bands on the autoradiograph. Looking back I should have got more quickly to the realization that the gel was permeable. When I was a child I became the favorite of my first teacher at primary school, Mrs Laycock. In those days the way we copied documents was to write a master copy in a thick violet-colored ink on a shiny highly carded paper. This was then laid on a tray of evil-smelling gel so that the ink soaked into the gel and copies could be made by laying blank sheets onto the gel to soak out the impression of the ink. I became very familiar with this method as Mrs Laycock employed me to use it to make copies of examination papers. So I learned a great deal, and I wonder if there was an echo of this experience at the light-bulb moment. I did not use the method much myself. Peter and I were beaten to the 5S genes by Don Brown, who used density gradient centrifugation to isolate the genes [6]. He went on to identify the region of the genes that control expression. Ironically, the control regions lie within the transcribed part of the gene—the very region that Peter Ford and I had sequenced to show the difference between somatic and oocyte RNAs! Nevertheless, the method caught on. And it was in widespread use long before it was published in the Journal of Molecular Biology. My first attempt at publication was rejected because it was a “methods paper” and so I had to spend a year or so gathering some more biologically relevant data before it could be accepted. However, it had already been disseminated informally. Late in 1973, we had a visit from Mike Matthews from Cold Spring Harbor Laboratory. I showed him my early results and he asked if he could have the protocol, which I sketched on a scrap of paper. Visitors to CSHL saw his work and in turn asked for the protocol. Mike asked my permission to pass it on, which I agreed provided

The Early Days of Blotting

3

that they were told the origin of the method. This probably explains why my name became so firmly a part of the method’s label. I was pleased and amused by the Northern (see Chapter 4) and Western blots (see Chapters 2 and 3) which quickly followed. References 1. Ford PJ, Southern EM (1973) Different sequences for 5S RNA in kidney cells and ovaries of Xenopus laevis. Nat New Biol 241:7–12 2. Kelly TJ, Smith HO (1970) A restriction enzyme from Hemophilus influenzae. II. J Mol Biol 51: 393–409 3. Shinnick TM, Lund E, Smithies O, Blattner FR (1975) Hybridization of labeled RNA to DNA in agarose gels. Nucleic Acids Res 10: 1911–1929

4. Chen CW, Thomas CA Jr (1980) A rapid twodimensional fractionation of DNA restriction fragments. Anal Biochem 101:339–341 5. Gillespie D, Spiegelman S (1965) A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol 12:829–842 6. Sakonju S, Bogenhagen DF, Brown DD (1980) A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5′ border of the region. Cell 19:13–25

The early days of blotting.

The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA...
137KB Sizes 1 Downloads 8 Views