Chromosome microtechnology: microdissection and microcloning

48

cloning techniqoes

Chromosome microdissection

microtechnology: 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 illumination pnth”J (Fig. 1). Sclcction of the laser system used dcpcnds on the rcquircmcnts ofthe specific application. Radiation damigc to DNA is minimized by avoiding the LN* of wavelengths close to the absorption maximum of DNA (-2h(! mn). UV laser wavclengths of -340 mn [c.g. the pulsed nitrogen laser (337.5 nm), the frcqucncy tripled NdYAG - ncodyniun: yttrium alunliniun~ garnet - laser (355 urn), and the tunable cxcimcr pumpbd dye laser] are sufficiently fnr away from this value, while still able to exert enough force for the precise ninnipulation of biological material. For the purposes of microdissecting 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 nanomctctY, is possibl@. Lasers used as optical tweezers for immobilizing biological material (i.e. optica! mpping) arc of 3 longer

TIBTECH JAN/FEE 1992 (VOL 10)

5ns

plJlS2 Pulsed UV-laser (Nitrogen iaser

x +

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.

wavclcngth - the NdYAG laser at 1Oh4 ml (infra-red) ir the light source of choice for this purpose. The working principle of the optical trap can be undcrstood by comparison with diclectrophorcsis, the techniquc 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 0 1992.

Elsevier Scmce

Publishers Ltd IUKI

49

clming techziques laser can be regarded as an inhomfield 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 cnvirontncnt 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 iiiicroinanipulation, procedures using lasers arc sterile, 2nd the power (ix. force escrtcd on chc biological tnamial) can bc easily controlled. Figure 2 shows the USCof a laser ttticrobeam and optical trap for microdissecting the tip of a chromosome in suspension and for iso!ating the ri+tctcd scgmcnc without any rncchanical COitWCt!‘_ of a NdYAG

ogcnous

A

clcctric

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 frlLgxcnts _ is known. cay1the 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 mapping and sequencing DNA. Fragnlen:s derived by frequcnt-cutter restriction cndonuclcase clcavagc can be amplified by PCR and cloned into vectors: chc typical 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 tmcgabascpair sized regions of the human genonx can bc obtaincd. Mapping of still larger strctchcs im~olvcs analysing uncloned gcnotmic DNA - a process which has bccotme fxsiblc through the dcveloprrient ofrarecutter restriction cndonuclcascs (i.e. cnzymcs with relatively few cleavage sites) aud PFGE (pulsed-field gel elcctrophorcsis). The bcrderlinr between iragtmcnts which can be analyscd by tmolecular biology techniques and those visible under the microscope arc’ fragtments in the region of 10 Mb. If DNA fragtments 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

w.

B

c

‘t

Figure 2 Microdissection of a chromosome and transport of the dissected chromosome fragment: (A) two chrcmosomes before microdissection; 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 scqucncing the genotmc, rcprcscnts an cttortnous task, it woL:!d bc &cirnt and realistic with the \vorkload hprcad ainong many laboratotics.

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TIBlECHJAN,~EB 1992 lVOi 101

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

100 chroniosonic segnicnts can bc prcparcd”‘,“. On Unfortunately, such n stmightfonvard, top-down npprmcli is not yet possible, bccausc microdisscction &is basis, PCl<-based microamplification is sup&or to microcloning. Howcvcr, when characteristics ofthc provides only a few-hundred fcintogranis to a few picogmns of DNA, resulting in inc&icnt cloning of libraries arc looked at in d&l, it bccomcs apparent that the method ofchoicc drpcnds on the application. the disscctcd region. Two alternative npproachcs can bc used to tncklc this problcni: microaiiiplific;ltioi7, Microlibrnrics must bc charnctcrized with regard tc whcrcby thr diF,cc,+cd fkrgmciit is aniplificd by PCR insert size, dcgrcc of redundancy, and whcthcr the and subscqucntly cloncd’~~‘~~;or n~icrocloning, whcrcclo~ics contain rcpctitivc :;r siuglc- or low-copy scqucnccs. For a microlibrary to bc useful ns n chroby the dissected fragment is cstractcd, digcstcd and library, it needs to provide ligated into the cloning vector in a volume of only 3 mosomc-scgmcnt-specific few iinnolitrcs14. Only by thcsc tcchniqucs can submaximal rrprcscntativc coverage of the cloned scgstnnti;ll numbers of DNA clones rcprcsenting the mcnt, with minimal redundancy (i.e. identical clones majority of the disscctcd fragment bc obtnincd 6om in :hc library). PCR-based 2nicrollmplific~ltion is pnrsuch minute amounts of DNA. titularly prone to gcncrating redundant clones, since. Many current tcchniqucs used for xxcning nnd in lntcr nmplification cycles, most of the template characterizing clones in YAC or cosmid libraries material is dcrivcd from previous amplifications, and rely on the use of hybridization probes dcrivcd from a minor proportion of the niolcculcs will not hnvc spccitic subct~roi~iosonial regions. Howcvcr. a sufparticipated in carlicr amplification rcnctions - such ficieut number ofsuch probes is available for only very scqucnccs have a lower probability of being cloned. few regions of the humnn gcnomc. Such probes have It is obvious that librnrics containing thousands of been used for saturation mapping with YAC cloncs~~clones amlot bc chnrxtcrizcd in their cntircty. Thus. of 1.5 Mb of thelong arm of humnn cl~ro~noso~t~c 7. to dctcrminc the lcvcl of rcdundnncy. 3 saniplc ‘Microlibrarics’ of lnr!;r number of microcloncs can (usually 5040 probes) is analyccd. Statistically, such provide a comp;lrativcly large numbers of probes of snmplcs arc likely to bc non-redundant, but they arc any dcfincd rcgh of the gcnomc and thus may lcad not rcprcscntativc of the whole library. (This can bc to ;i signifkmt improvcnient in saturation mapping illustrated simply with a deck of 32 playing cards, distcchniqucs. rcgrding the ditfercnt colours. The deck hns 3 x 8 cards of difkrcnt values, corresponding to four copies MicroampliJiration versus microcloning of tight difkrcnt DNA ~~dxulcs. Thr deck is thcrcWith PC11 microamplificati~~i, n librnry of up to fort &$ly rcduudanr, since one can rctnovc 75% of 700000 DNA clones with inserts a kw-hundred bnscs the cards and still retain one copy of each of alltight in size has been prcpnrcd from ns few as 30 chromodiftkcnt-value cards. Statistially, a sample of three some scgmcnts’“. With microcloning of DNA into cards ~111, in most casts, give three different-value lambda-phage vectors, about 5011 DNA pro&s per cards, thus lading to the crroncous conclusion that @TECH JAN/FEE 1992 (VOL 10)

51

clonirg techraiques :here

is

malo~y

s ncarlv

wn6.j

redundancy.

nc that

One

could

argue

in this cards clones is ICSFthan

the sample size is too small. but 3/31

10?4, whereas

50/700000

inosorncs

from this analobsg is that, for systcmttic 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. Tht

Thcrc is still a long way to go bcforc suc11an approxh for saturation sequencing is practicable, but lxgc-scale. laser-based microdisscction and manipulntjon ofchro-

mcssagc

to proxidc

in thi< mcgascquencing

an iinportant

approach.

References 1 2 3

4 5 6

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 laserupporccd chromosome scgmcnt prcpaiation is valulblc in gcncraring hundreds ofchromoson~c segmcnrs gcnomic region, enabling the prcp‘ram a d&led 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 hvbridization’ “, for which kilobn.sc>ligonuclcotidc , izcd DNA fragmcnfs as probes appear to bc optimal, s adopecd as a fcasiblc approach. Systematic scqucncng of GO00 overlapping microcloncs \vould provide I twofold covcragc ofn 10 Mb chromosomnl scgmcnt; .c. a tyl*: of shotgun approach would be possible.

has the potential

step forward

7 8 9

11 12 13 ; ?

IS 16

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