although the risk is still higher than in the general population. BRCA2 also differs from BRCA 2 in being associated with a considerably greater risk of breast cancer in males. However, since the absolute risk of males in these families developing breast cancer is still small (approxi- mately 5% by age 70), it is likely that many families with BRCA2mutations will be characterized by female breast cancer only (Fig. 1).

The difference in risk of breast cancer in males conferred by mutation in BRCAl and BRCA2 is interesting from a mechanistic per- spective. The male and female breasts follow the same programme of em- bryological development and ap- pear identical through childhood until puberty. At puberty, changes in levels of circulating sex steroids induce proliferation of the cellular components of the breast and de- velopment of lobules in females. In males, a predominance of androgenic steroids results in much lower pro- liferative activity and absence of lobules. These homlonal changes and their biological sequelae also de- termine the greatly different rates of

COMMENT

breast cancer between the two sexes. Since it seems that there is little excess risk of breast cancer in males cartying mutations in BRCAl, it would appear that the protection conferred by androgens in these men is essen- tially intact. By contrast, in male carriers of BRCAZ mutations protec- tion has, at least partially, been lost. We may therefore speculate that the BRCAl and BRCA2 proteins occupy rather different positions with respect to sex hormone regulation.

Other breast cancer genes? A new positional cloning race has

started for isolating BRCA2 - con- veniently, just as the first runner in the race to clone BRCAl has broken the winning tape. Advances in tech- nology may mean, however, that this race will be more sprint than marathon. Meanwhile, it appears that there are yet more breast cancer genes to be found. Certain families have a genetic predisposition to the disease that is not linked to either BRCAl or BRCA2. Moreover, it remains to be determined whether genes other than BRCAl or BRCAZ may have an equal, or perhaps

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greater, contribution to the overall incidence of breast cancer: although mutation of such genes might confer a lower excess risk of breast cancer than BRCAI or BRCA2 mutations, such aberrations may be more common within the population as a whole.

Acknowledgement We thank the Cancer Research

Campaign for their support.

References Higginson, J., Muir, C.S. and Munoz, N. (1992) in Human Cancer: Epidemiology and Environmental Causes, pp. 377-387, Cambridge University Press Easton, D.F., Ford, D. and Peto, J. (1993) Cancer Surv. 18, 95-113 Hall, J.M. et al. (1990) Science 250, 1684-1689 Narod, S. et al. (1991) Lancet 338, 82-83 Easton, D.F. et al. (1993) Am. J. Hum. Genet. 52, 678-701 Miki, Y. et al. (1‘994) Science 266, C&71 Futredl, P.A. et al. (1994) Science 266, 12~122 Stratton, M.R. et al. (1994) Nature Genet.7, 103-107 Wooster, R. et al. (1994) Science 265, 208S2090

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(A) (‘3

a bcdef g

FIGWRE 2. (A) Lineage-marked blastomeres. Phase-contrast micrograph of (a) zona-free early blastocyst; (b) isolated KM; (c) TE cell cluster labelled with HTC-concanavalin A; (d) fluorescence micrograph of TE cell cluster shown in cc>; <e) phase-contrast micrograph of single cells from blastocyst in fc,d) labelled with HTC-concanavalin A, CD fluorescence micrograph showing iabelled TE cell (left) and unlabelled KM cell (right). Scale bar = 10 brn. (3) RT-cDNA amptification of MAP-purified mRNA from: (b,c) blastocysr; (d) isolated KM; (e) TE four-cell cluster; Cf) single KM cell; (g) single TE cell, using (c-gt either DSC2 primers (top) or E-cadherm primers (bottom), and (b) both primer sets simultaneously. Lane (a) contains IDO bp DNA ladder. Transcripts are detected in single cells, and one transcript only is found in KM cells. Products are checked by DNA sequencing and a positive E-cadherin control is always used.

amplification of extracted mRNA allows the detection of two specific mRNA transcripts in single cells from blastocysts, as wel! as in ah preirnplantation stages, including eggs and isolated inner cell masses (KMs), The combination of embryo manipulation and cDNA amplification provides a resolution of mKNA detection not previously achieved in studies of preimplantation development, and permits analysis of the earliest transcriptional events underlying differentiation within individual embryos. Et ehminates prob- lems caused by differences in the timing of development between embryos and reduces the number of embryos required for molecular studies; the technique can even be applied to analysis of human development, where availability of material is very limited.

I Zona-free early blastocysts are obtained as described previouslya. ICMs are isolated by complement-mediated lysis of the outer trophectoderm (‘LB) layer3 (irnmunosurgery) and incubated in DNase-free RNase (Boehtinger ~eim) (10 wg ml-1 in M2+BSA embryo culture medium, 20 mm). Embryos are then washed in ML+BSA to remove any cc&ami- nating RNAs from TP lysis.

2 To collect single TB and KM cells, blastocysts are treated with PITC-concanavalin A (Sigma) (1 mg ml-* in MZ+BSA,

volume (~5 of M2+BSA. P&y(A)* RNA is prepared by lys

inhibitor (Pharmacia) and 200 U Superscript I RNaseH-free reverse transcriptase in

5 reaction is incubated for 10 min at room temperature, 45 min at 37% and 5 min at 95% Two 10 ~1 aliquots are placed in fresh tubes for cDNA amplification: one is used for assay, the other for a The volume of each is made up to 35 @I by addmg 3.5 pl X 10 Vent buffer (New England f&labs), and MilliQ water (Mihipore). Paise priming is eliminated using ‘hot start’ with Amp&wax K% gems 1 LJ Vent polymerase (New England Biolabs) in 15 ~1 of X 1 Vent buffer is added above the WF after i fication is carried out for 25 cycles as follows: 95%, 30 s; 53”C, 60 s; 72%, 40 s,

6 A second DNA-ampliication reaction is set up as above, using the inner nested primers and 2 ~1 of the &at arn~~~~o~ reaction as template. Amplification is carried out for 25 cycles in the case of whole embryos or LCMs; nr 3Q e@es for single cells, as follows: 95*C, 30 s; 53”C, 60 s; 72Y, 40 s.

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ACKNOWLEDGEMENT This work was funded by a grant from the Wellcome Trust to T.P.F.

REFXRENCE~ 1 Sheardown, S. (lY)Z) Trends Genet. 8, 124 2 Fleming, T.P., Garrod, D.R. and Elsmore, A.J. (1991) Deue6opment 112, 3 Chisholm, J.C. et al. (1985) J. Embryol. E@. Molphol. 86, 311-336 4 Buxton, R.S. et al. (1994) Genomics 21, 510-516 5 Lorimer, J.E. et al. Mol. Menzb. Biol. (in press) 6 Nagafuchi, A. et al. (1987) Nature 329, 341-343

Contributed by Jane Collins and Tom Fleming, Department of Biology Southampton, UK SO9 .?TU

Many high-temperature DNA polymerases produce PCR products with a 3’-dA overhang. which prevents blunt-end iigation of the products to a vector DNA.‘This problem may be overcome by (1) ‘polishing’ the PCR ends using ‘I4 DNA polymerase in the presence of dNTP; (2) using commer- cially available cloning kits that contain a preprocessed vector DNA with a 3’-dT overhang; (3) using polymerases, e.g. pfu DNA polymemse, that produce blunt-ended PCR products. Here, we describe two very simple methods for cloning PCR products generated by Taq DNA polymerase.

The first method (see Fig. 1) takes advantage of the fact that some restriction enzymes generate a 3’ overhang with a terminal dT, which can base-pair with the 3’-d&overhang

Single-step direct cloning of PCR products

vector ---GAGCT NNNNNNNA c--- - - - c ANNNNNNN TCGAG---Vector

---GAGCTNNNNNNNA C--- --_ c ANNNNNNNTCGAG---

1 1 method 1

Sad Sacl+EcoRI -.-___

method 2

Bamtil +Pstl

of the PCR products; examples of such enzymes are Sac1 (GAW’PC), PvuI (CGATKG), AatII (GACGTW), and PacI (TTAATTAAA). These enzymes produce two- or four- nucleotide 3’ overhangs (shown in bold typeface). The only requirement for ligation is that the PCR product must con- 3.66 kb tain a 5’ phosphate. In a typical experiment, using a kinased 2.96 kb

primer pair, the PCR product generated using Taq DNA polymerase (719 bp) was ligated with SacI-digested pBlue- 0.72 kb script II SK(+) (Stratagene). After transformation into E. coli DHSa, 20% of colonies that grew on LB plates containing ’ X-Gal, IPTG and ampicillin were white. Of these, 83% were correct recombinants. Since both primers used in this exper- FIGURJS 1. Cloning strategy for method 1 (top) and restriction iment started with a 5’-dC, it was expected that Sac1 sites analysis of recombinant DNA (bottom). Top: Vector DNA, flanking the insert would be regenerated. However, instead linearized by SacI, is ligated with the PCR product containing of releasing the insert, Sac1 digestion linearized the plasmid a 5’ phosphate and a 3’-dA overhang, which can base-pair in all of five randomly picked recombinants (Fig. 1 bottom: with the terminal dT of the 3’ overhang of the vector DNA. 3.68 kb band, lanes l-5), indicating that only one Sac1 site Bottom: Five randomly picked recombinant DN.4.s from either was regenerated. When the plasmids were digested with method are digested with various restriction enzymes as both Sac1 and EcoRI (Fig. 1 bottom: lanes 6-101, all five indicated. Sizes of fragments are indicated to the right. recombinants produced the 719 bp insert plus the 2.96 kb Mt ‘stEI1digested phage A DNA‘ vector band, indicating that in all cases tested, the SacI site closer to the EcoRI site was not regenerated. The reason for this is not clear. A single-tube reaction including both cleavage and ligation can be performed using uncut vector DNA, provided that the PCR product contains no cutting sites for the restriction enzyme used and ligation does not regenerate the cutting sites. This method has the advantages of bypassing the need to process PCR products before ligation and needs no expensive commercial kits. Although other 3’ overhangs are also produced by Taq DNA polymerasel, it will not interfere with the ligation, because the PCR products with a 3’-dA overhang are always present and most OF the time are the major products. We have been using this method for cloning of many other PCR products with great success, indicating its usefulness in general.

The second method entails a one-tube ligation in which digestion of vector DNA, polishing of PCR DNA ends and blunt-end ligation are accomplished in a single step. PCR products are incubated with the uncut vector pI3luescript II SK(+) DNA in the presence of 2 U SmaI, 1 U T4 DNA polymerase, 0.03 mM dNTP, 3 U T4 DNA ligase, 1 mM A’I‘P, in the ligation buffer in one tube. The 3’-exonuclease activity of T4 DNA polymerase polishes the ends of the PCR product ln the presence of dNTP. Small digests the vector DNA and forces the insert into the vector by continuously cutting self-ligated vector DNA. In a typical experiment, the PCR product is generated using the same primer pair as above, but is not phosphorylated. After tmnsformation into DH%, about 10% colonies were white and 70% of these contained the correct inserts, When DNA from five randomly picked recombinant

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0 1995 El\cvierS<lmrc LidOlh8 9525~95/$W 50 7