48

cloning techniqoes

Chromosome microtechnology: microdissection and microcloning

Karl Otto Greulich The physical microdissection of chromosomes and subsequent microcloning of

dissected fragments is enabling the generation of very large numbers of cloned

unique sequenw frcm defined chromosomal regions. In addition to use in con-

structing region-specific libraries of the entire human genome and providing probes

for mapping and sequencing purposes, such chromosome microtechno!ogy should

facilitate the search for disease-associated genes in defined chromosome regions.

Focused UV lnscr microbcams arc highly suited for precision manipulation of chromosomes, able to rcplacc mechanical micronccdlcs for I7licrodisscction’, and also to be used 3s ukrafinc rwcczcrs2. They have bczn used for the microdissection of chromosomes’. &ion ofpnirs ofccllc’, and microinjcction ofmatcrial into ~11s~~ and subccllular structur&. To optimize the spatial accuracy of the manipulation, lacers arc coupled into a microscope, usually through the illumi- nation pnth”J (Fig. 1). Sclcction of the laser system used dcpcnds on the rcquircmcnts ofthe specific appli- cation. Radiation damigc to DNA is minimized by avoiding the LN* of wavelengths close to the absorp- tion maximum of DNA (-2h(! mn). UV laser wavc- lengths of -340 mn [c.g. the pulsed nitrogen laser (337.5 nm), the frcqucncy tripled NdYAG - nco- dyniun: yttrium alunliniun~ garnet - laser (355 urn), and the tunable cxcimcr pumpbd dye laser] are suf- ficiently fnr away from this value, while still able to exert enough force for the precise ninnipulation of biological material. For the purposes of microdissect- ing chromosomes, the photon-density of the pulsed UV laser microbcam is 6r above the threshold for burning or boiling, and thus ablation of chromosomal material with an accuracy of a few hundred nano- mctctY, is possibl@.

5ns plJlS2

Pulsed UV-laser x (Nitrogen iaser + P k=337nm) High peak power

I/

Continuous IF&laser (Kd YAG laser - h=l064nm) Moderate power

L

Figure 1 Laser microbeam and optical trap: the pulsed nitrogen laser with an ultraviolet (UV) wavelength of 337 nm is used ?or microdissection of chromosomes. The continuous NdYAG laser with an infrared (IR) wavelength of 1064 nm is used for transpori of microdissected chromosome segments. Both lasers are coupled into a Zeiss IM35 microscope via the epifluorescence illumination path.

Lasers used as optical tweezers for immobilizing bio- logical material (i.e. optica! mpping) arc of 3 longer

wavclcngth - the NdYAG laser at 1 Oh4 ml (infra-red) ir the light source of choice for this purpose. The working principle of the optical trap can be undcr- stood by comparison with diclectrophorcsis, the tech- niquc used to collect cells prcccding clectrofusion. This technique uses the fact that, in inhomogenous electric fields, dielectric objects move towards the point ofhighcst &Id strength. Since the focused light

TIBTECH JAN/FEE 1992 (VOL 10) 0 1992. Elsevier Scmce Publishers Ltd IUKI

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49

clming techziques

of a NdYAG laser can be regarded as an inhom- ogcnous clcctric field with the hig!lesc field strcngh exactly in the focus, chromosonm arc pulled towards the focus whcrc they cau bc restrained’. Relative motion bctwccn the fiscd parciclc and the cnviron- tncnt can bc generntcd by moving the XY stage of the niicroscopc. FOT csamplc, chroinosot7cs can bc moved”’ in the depth ofa ccl:. Since light, rather than incchanical contact, c,xcrts the force for iiiicroina- nipulation, procedures using lasers arc sterile, 2nd the power (ix. force escrtcd on chc biological tnamial) can bc easily controlled. Figure 2 shows the USC of a laser ttticrobeam and optical trap for microdissecting the tip of a chromosome in suspension and for iso!at- ing the ri+tctcd scgmcnc without any rncchanical COitWCt! ‘_

The role of microdissection in the hierarchy of genome analysis techniques

The USC of a laser microbcatm and an optical trap pcnmits ihc preparation ofhundrcds offragncnts from a chroniosonial prcpai.- tion in only 3 few hours, thus providing a new tool in the analysis of the human gcnonic for rapid and convenient isolaeion of specific chroimosoimal regions. Current DNA-sequencing tcchniqucs arc liniitcd to analysis of molecules below 1 kb in size. The three gigabascs (Gb) of the human genome tmust thcrcforc bc disscctcd into tnorc than 3 x 106 fragmcntc for scyuencing, and only when the linear order ofthcsc frl _ L gxcnts is known. cay1 the cntirc gcnotnic scqucncc be assctnblcd. At present. thcrc is no direct way of obtaining such an ordcrcc! collection of fragnlcnts and the long-range scqucncc is derived through a hierarchical setics of techi)iqucs for map- ping and sequencing DNA. Fragnlen:s derived by fre- qucnt-cutter restriction cndonuclcase clcavagc can be amplified by PCR and cloned into vectors: chc typi- cal six of the resulting cloned DNA is 0.3-0.4 kb. The next orders of tmagnitudc in six of the cloud DNA can bc obtained by microcloning into lambda phagcs (3 kb), costmids (50 kb) or yeast artificial chromosomes (YACs) (300-500 kb). By overlapping groups of clones that collcctivcly span a particular chromosornal region (contigs), stqucnce for tmcgabasc- pair sized regions of the human genonx can bc ob- taincd. Mapping of still larger strctchcs im~olvcs analysing uncloned gcnotmic DNA - a process which has bccotme fxsiblc through the dcveloprrient ofrare- cutter restriction cndonuclcascs (i.e. cnzymcs with relatively few cleavage sites) aud PFGE (pulsed-field gel elcctrophorcsis). The bcrderlinr between irag- tmcnts which can be analyscd by tmolecular biology techniques and those visible under the microscope arc’ fragtments in the region of 10 Mb. If DNA frag- tments can bc isolated from such visible chrotmosome scgtncnts by microdisscction and rhen analyscd. the spatial accuracy of assigning the scqucncc to specific chromosotmal regions is of the order of a few tens of tmcgabascs. This can bc further iniproved by aligning clones based on overlapping sequences. ldcally, rmicrodissrcced chrotmosnnte scgtmc!:ts \vould

A w.

B

c

‘t

Figure 2 Microdissection of a chromosome and transport of the dissected chromosome fragment: (A) two chrcmosomes before micro- dissection; 0 the tip ?f one chromosome is microdissected with the nitrogen laser; (C) the microdissected fragment is fixed in the focus of the NdYAG laser, and by moving the XY stage, the fragment and the rest of ihe chromosome are separated. The isolated chromosome segment can be kept in the focus, which thus acts as a sterile, wallfree microvessel.

be usd for the construction of.3 contiguous library of a few hundred YAC clones, and thrn cnc!: YAC cionc could bs the starting point for the construction of a few hundred latnbda clones \vhich tttighC be dircccly scqucnccd or used to order a library of TCR cloncz. Although this approach, as all approaches to scqucnc- ing the genotmc, rcprcscnts an cttortnous task, it woL:!d bc &cirnt and realistic with the \vorkload hprcad ainong many laboratotics.

__~_._ _ TIBlECHJAN,~EB 1992 lVOi 101

“When you have quite finished, Wilkins, we have a chromosome to dissect!” J

Unfortunately, such n stmightfonvard, top-down 100 chroniosonic segnicnts can bc prcparcd”‘,“. On npprmcli is not yet possible, bccausc microdisscction provides only a few-hundred fcintogranis to a few picogmns of DNA, resulting in inc&icnt cloning of the disscctcd region. Two alternative npproachcs can bc used to tncklc this problcni: microaiiiplific;ltioi7, whcrcby thr diF,cc,+cd fkrgmciit is aniplificd by PCR and subscqucntly cloncd’~~‘~~; or n~icrocloning, whcrc- by the dissected fragment is cstractcd, digcstcd and ligated into the cloning vector in a volume of only 3 few iinnolitrcs14. Only by thcsc tcchniqucs can sub- stnnti;ll numbers of DNA clones rcprcsenting the majority of the disscctcd fragment bc obtnincd 6om such minute amounts of DNA.

Many current tcchniqucs used for xxcning nnd characterizing clones in YAC or cosmid libraries rely on the use of hybridization probes dcrivcd from spccitic subct~roi~iosonial regions. Howcvcr. a suf- ficieut number ofsuch probes is available for only very few regions of the humnn gcnomc. Such probes have been used for saturation mapping with YAC cloncs~~ of 1.5 Mb of the long arm of humnn cl~ro~noso~t~c 7. ‘Microlibrarics’ of lnr!;r number of microcloncs can provide a comp;lrativcly large numbers of probes of any dcfincd rcgh of the gcnomc and thus may lcad to ;i signifkmt improvcnient in saturation mapping tcchniqucs.

MicroampliJiration versus microcloning With PC11 microamplificati~~i, n librnry of up to

700000 DNA clones with inserts a kw-hundred bnscs in size has been prcpnrcd from ns few as 30 chromo- some scgmcnts’“. With microcloning of DNA into lambda-phage vectors, about 5011 DNA pro&s per

&is basis, PCl<-based microamplification is sup&or to microcloning. Howcvcr, when characteristics ofthc libraries arc looked at in d&l, it bccomcs apparent that the method ofchoicc drpcnds on the application. Microlibrnrics must bc charnctcrized with regard tc insert size, dcgrcc of redundancy, and whcthcr the clo~ics contain rcpctitivc :;r siuglc- or low-copy scqucnccs. For a microlibrary to bc useful ns n chro- mosomc-scgmcnt-specific library, it needs to provide maximal rrprcscntativc coverage of the cloned scg- mcnt, with minimal redundancy (i.e. identical clones in :hc library). PCR-based 2nicrollmplific~ltion is pnr- titularly prone to gcncrating redundant clones, since. in lntcr nmplification cycles, most of the template material is dcrivcd from previous amplifications, and a minor proportion of the niolcculcs will not hnvc participated in carlicr amplification rcnctions - such scqucnccs have a lower probability of being cloned.

It is obvious that librnrics containing thousands of clones amlot bc chnrxtcrizcd in their cntircty. Thus. to dctcrminc the lcvcl of rcdundnncy. 3 saniplc (usually 5040 probes) is analyccd. Statistically, such snmplcs arc likely to bc non-redundant, but they arc not rcprcscntativc of the whole library. (This can bc illustrated simply with a deck of 32 playing cards, dis- rcgrding the ditfercnt colours. The deck hns 3 x 8 cards of difkrcnt values, corresponding to four copies of tight difkrcnt DNA ~~dxulcs. Thr deck is thcrc- fort &$ly rcduudanr, since one can rctnovc 75% of the cards and still retain one copy of each of all tight diftkcnt-value cards. Statistially, a sample of three cards ~111, in most casts, give three different-value cards, thus lading to the crroncous conclusion that

@TECH JAN/FEE 1992 (VOL 10)

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clonirg techraiques

:here is nc redundancy. One could argue in this malo~y that the sample size is too small. but 3/31 cards s ncarlv 10?4, whereas 50/700000 clones is ICSF than wn6.j

Tht mcssagc from this analobsg is that, for systcm- ttic constmctioii of 3 chromosornc-rc~ion-specific ibrary, thr classical microcloning approach is more tppropriatc, since by its nmchanisnl, it gcncrates css redundancy. Howcvcr, if scvcral ccns of DNA xobcs of small sizr (e.g. 0241.3 kb) arc required for x-obing, for cxamplc, a YAC library, I’CR-based iiicroartiplification is the method of choice since Jinost all of the DNA probes sclcctcd will be diffcr- :nt cvcn ifthcre is a high lcvcl of redundancy within hc whole library.

qpplication of laser microdissection and optical .rapping to megasequencing

With PCR arnplitication techniques. a sin& :hronlosomc scgmcnt may bc sufficient to gcncratc I rcasonablc number of DNA clones. With good cpnmtion of the mctaphasc chri)mosomcs, the high Icgrcc of precision with which the laser can bc argctcd allows material to bc obtained t?om only one ,fthe sister chromatids. III addition, the speed of laser- upporccd chromosome scgmcnt prcpaiation is valu- lblc in gcncraring hundreds ofchromoson~c segmcnrs ‘ram a d&led gcnomic region, enabling the prcp- motion of a large number of kilobasc-sized molecules.

This aspect could gain significantly in importance if hc concept ofscquencing large amounts of DNA by >ligonuclcotidc , hvbridization’“, for which kilobn.sc- izcd DNA fragmcnfs as probes appear to bc optimal, s adopecd as a fcasiblc approach. Systematic scqucnc-

ng of GO00 overlapping microcloncs \vould provide I twofold covcragc ofn 10 Mb chromosomnl scgmcnt; .c. a tyl*: of shotgun approach would be possible.

Thcrc is still a long way to go bcforc suc11 an approxh for saturation sequencing is practicable, but lxgc-scale. laser-based microdisscction and manipulntjon ofchro- inosorncs has the potential to proxidc an iinportant step forward in thi< mcgascquencing approach.

References 1

2 3

4

5

6

7 8

9

11

12

13 ; ?

IS 16

17

18

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