3 .2 .3 .1
KpnllBssHW
digestsD N A from cosmid D1020 was digested with Kpnl and t o H II , the fragments were separated on agarose gels and those containing the ends of the human insert D N A , identified by their hybridisation to vector D N A , were excised and purified. These fragments were radiolabelled, preannealed for 3h and hybridised to membranes containing human D N A digested with either EcoRl or Hindlll. After hybridisation these membranes
were washed to high stringency. Despite preannealing, these fragments showed
hybridisation to all parts of the tracks containing the human D N A and were, therefore, highly repetitive and of little use in screening the cosmid library (results not shown).
3 .2 .3 .2 Riboprobes
Fig. 3.3 Physical mapping of the DXS265 sequence
00 < 00 < oo < la ^ oo X < ^ X 00 10 < hc s S * ' * : i “ • -14.8 ^ 1 3 . 4 , 0 ^ # # l & 8 6.2 - 5. 3 26 .2 — fc. 4— : ' l 0 . 6 — ► # 6.2 —► 4k 4 — , DXS26 5 DXS178 LoristDNA from cosmid D0590 was digested with the enzymes shown (K = Kpnl, B = A = Nael),
separated by PGFE, using conditions: 190V, 3s switch time, 16h; and hybridised with probes specific for the DXS 178 and DXS265 loci and the Lorist vector. Hindlll digested lambda DNA and lambda concatamers were used as size markers. Fragment sizes are in kb.
o f Lorist B (Cross and Little, 1986). This vector contains the viral R NA polymerase promoters T7 and SP6 in such an orientation that they face into the human insert from
opposite ends of the vector sequence. RNA transcripts can be made from these
promoters such that they contain the ends of the human insert. The riboprobe system utilizes these promoters to produce RNA probes radiolabelled to high specific activity for use in hybridisation to D N A .
Cosmid clone E03167 was used for the preparation of riboprobes as it contained different D N A at the ends as compared to D1020, which had already been shown to be repetitive. The cosmid template was digested with restriction enzymes prior to the in vitro transcription reaction so that very long transcripts were not produced. For the T7 reaction, the 5.6kb Kpnl/BssUll fragment was used (Fig. 3.2) as this contained the T7 promoter and sufficient human insert to produce a probe of suitable length. For the SP6
reaction the corresponding BssHll/Kpnl fragment containing the opposite end o f the human insert would have produced too long a transcript. EcoBJ was used as this cut vector D N A sufficiently far from the SP6 promoter to allow efficient transcription and
cuts human D N A sufficiently frequently to make it unlikely that a very long transcript would result.
The in vitro transcription reactions were carried out on both the cosmid templates described above and on control templates (Promega). The percentage incorporation of radioactivity into the transcripts prepared from the cosmids was low in comparison to the control template. 1.65% compared to 95% for the SP6 promoter and 13% compared to
94% for the T7 promoter. The transcripts were, however, of the expected length and were o f comparable specific activity to the control templates indicating that they had been produced in smaller quantity. They were hybridised to EcoRI or H ind lll digested human D N A . Examination of the autoradiographs showed that the tracks had a high activity along their lengths, indicating that the probes were repetitive in nature (results not shown).
3 .2 .3 .3 Use of small
Kpn\
fragmentsAs the ends of the D N A inserts in these cosmid clones had proved to be repetitive, it was decided to test the small fragments produced by Kpnl digestion o f clone E03167 for suitability as probes. As these fragments were only a small fraction of the
total cosmid insert, preparing them from the cosmid each time required the use of large quantities of cosmid D N A , so these fragments were first subcloned into the plasmid
vector pSP72. Plasmids containing each of the fragments were radiolabelled and
hybridised to human D N A to test for the presence of repetitive sequences. F I (Fig. 3.2) was completely repetitive in nature but F II and F IIA (Fig. 3.2) both produced specific hybridisation patterns against a high background. These were investigated further.
In order to identify less repetitive fragments from these clones, plasmids containing F II (p F II) and F IIA (p F IIA ) were digested with Kpnl to excise the insert and then with Taql, EcoRI, //m d lll, BaniRl, B g/II, H aelll, or Pstl. Digestion products were blotted onto nylon membranes and hybridised with total human sonicated D N A , and with D N A from the pSP72 vector. Absence of hybridisation to either o f these probes was taken as an indication that a fragment was both non-repetitive, and from the human insert. Only B g /II, H inùlll and Taql cut either o f the inserts, and only a 0.4kb BgWKpnl fragment of the p F II plasmid (F II/B ) and a 0.2kb Hindlll!Kpnl fragment of the pFIIA plasmid (F IIA /H ) showed no hybridisation with either of the probes.
The F II/B fragment was hybridised to human genomic D N A digested with Taql or Kpnl. A hybridisation pattern of three strongly hybridising species with Taql and four strongly hybridising species with Kpnl (Fig. 3.4) was seen. The strongly hybridising bands were all larger than the size of the probe. The human insert of the p F II probe had no sites for Kpnl, and only one site for Taql, thus the probe could not be legitimately hybridising to all of the fragments seen. There were two possible explanations for this:
■ the D N A on the membranes was only partially digested. ■ the probe being used represented a mildly repetitive element.
The F IIA /H fragment hybridised to a single fragment on both Kpnl and BamYlll digested human genomic D N A (Fig. 3.4) although there was still a large amount of background hybridisation, particularly to the partially digested D N A at the top of the lanes in Fig 3.4. Both fragments F II/B and F IIA /H were used sequentially, to rescreen the cosmid library. The fragments were preannealed with human D N A using the PERT method prior to use. The F II/B fragment hybridised to a large number of colonies with strong hybridisation to more than 100. This was, therefore, a repetitive element and was not used further. The F IIA /H fragment showed hybridisation to 10 discrete colonies (9 on duplicate membranes). These were requested from IC R F, London.