PCR amplification techniques for chromosome walking

44 clmirzg

technipes

PCR amplification techniquesfor chromosome walking AndrkRosenthal ~ Amplifkkm

of DNA segments i/r r&o by 0.x polchain reaction WJ<)’ is 3n enzymatic for gcncrating miliions of identical copies of a rclativcly short IPNA molcculc in a few hocus without the riced for cmvc-ntional cloning tcchniqucs. It is n pcm ~~~ftll tcchniqiuc whi4 hns f&id numerous applications in biology :in~~ incdlcinc’-.‘. Typically, I’CR ainpliticationc LISC ;wo oligoynicrasc method

~~~~lcotidc prirncrs that hybridize to opposite strmds x-d :I thcrmoof a specific known I)NA fragment, scablc DNA polymcrm which cstalysr, the rtnd copyiiig rccuxion. The prinicrs arc oricrrxd srlch that clongatir: yrocecds inwnrds ;Icro.ss the region hcrwcur 9x5 two primers. Rcpctitivc cycles of. dcrr;rturiug prilncrs.

the newly synthccizcd strands, annealing the ,md DNA synthesis Icad to nn exponential

:;mpiificntion of the cargct I)NA fragment. Applications

of PCR

in ;1 dircctrd manner. Nor CJII it bc used for scqucntial ‘walking’ along chromoso1ml DNA. Scvcral new PCR-basrd amplification tcchniqucs have recently been dcvclopcd to ovcrcomc thcsc liruitntions: ??Attlpltjkation usittg wpetitive, random or degenerated prthevs One approach for amplit$ng unknown IINA

is lo prinicrs which m-c coniplcmcncnry to rcpctitivc clcmcnts which nrc dispersed throughout the gcnomc. In pnrticcllar, Aiu, Kpn or other intcrsperscd repetitive scquc~xxs jlK5) have been used to deign primers foi LISC cithcr WI their own for nnalysing YAC inserts (c.g. AILI-I’CR) or in combination with primers basrd on vector scqucnccs to isolate YAC rnds (Alu-vector YCR). IRS-IXR, cspccially Alu-I’CIL. can also bc cscd L>r fingerprinting cornplcs gcnomcs’ and is a very vrluablc tool for gcncrating new probes and scqucncc taggr Li sites (STSs) in region< poorly covered with 1)NA markers (SW I\. Anand, pp. 35-40 in this issue of TlU’IECl-I 1. tlCNA gxcs. which occur in nlultiple topics dispcrscd throqhm~t the gcnomcs of many spccics, could al&.0 bc a source for deriving CQIISCIISLIS use

primer scqucnces. l’Cl<,

has rcccntly

This ecchniquc, bcctl

dcscribcd

known 3s tONAfor fingerprinting primcrs of random

cukaryotic gcnom~s~~. Shorter scq~ncc or conlainiiig vqing amounts :Lte scqucncc

cm :&FObc used for this

oidcgcmx-

p~~qxxx

sequences - inverse PCR first introduced in 19887,x, involves digestion of gcnomic DNA with a suicablc restriction c~~zyrne that cuts the tcmplatc oucsidc the known locus. After circulnrizing the fagmolts by ??Rnalysingflataki~tg

Invcrsc

8

i’CA (I-PCII),

1392, Elsewe: Science Pubhshers Ltd (UK)

45

cloning techniques ligation. the u~~known IINA scgmcnt is then flnnkcd 011 both sides by tlrc knowr~ locus. Alnplification is carried out with two priincrs that hyhridizc to the ends ofthc known locus, but arc rcvcrscd in their dircctitril with rcspecc to their normal oricnt.~tion in I’CR (Fig:. 1). Invcrsc I’CIL Ius been used primarily for imllting the end rcqucnccs of YAC clones in total yeast ~cnoniic DNA “.“I l
known

-9. lenomic

locus.

_-AL_

DNA I cleave with

restriction

I

enzyme

A ---

l circularize

by

ligation

I

;Ikn

??An$iJicrltiorr techniques based on syrrthctic oligo cassettes Other mc:hodc IISC synthetic oligonuclcoticlc cmcttcs as a binding rcgi
I

exponentiai

two primers

PCR using

J Figure 1 Three steps II Inverse PCR”1: see text for details

cornplcsity of the gcnomic DNA used. l‘hc COIIIplcsity uf tlx g;cnomic DNA is dcfincci by the cize of the gnome undrr investigation. its orgnnizntion and structure (c.g. the number of cinglc-copy nnd rcpctitix~c scc~ucnccs). the nurnbcr of difi>rcnt IINA molcc!lles prrtcnt in a gi\fcn IINA prcp.uxtion. ;~nd other

b

1

2

(i) double-stranded cassette

known locus

m 7 2

{ii) single-strandeJ cassette

==EzCxx 4-1 2

(iii) panidlly cassette

single-stranded

with mismatch

I

I

I “;-

m

I PCR with locus-specific casseite-specilic

primer

primer 2

1

and

(iv)

partially sir alestranded cassette (Lectorotfe’ or ‘butble’)

t

-

I 7

Figure 2 (a) Amplification techniques using synthetic oligonucleotide cassettes 12-15: The specificity of the amplificaticn yeaction depends on the structure of the cassette and the con:slexity of the genomic DNA used. (b) Different c&sette designs. ii) This doublestranded CSSS&Z IS not sultable because primer 2 will bind to both ends of all restriction fragments carrying the cassette and initiate nonspecific amplification. Specific amplification between primer 1 and 2 is unlikely to occur. (iiHiv) These cassettes possess singlestranded portions to which primer 2 cannot initially hybridize. Only after the locus.specifir primer 1 has been extended in the first PCR cycle, primer 2 can bind and take part in the subsequent amplification cycles. Cassettes with mtsmatches~~ or ‘bubble’structufei4 have been introduced to prevent the ‘Lllingin’of the 3’ recessive strand. 7IBIECH JAN,FEB 1952 IVOi :2!

A

known locus

_-

genomic DNA 1

cl&vewith re8triciion enzyme

c

e--

I

ta

*denaWe *anneal prime: 1a ?? etiend primer 1a with sequenase

1 ______.____._..________---“-~..

I ligate linker to blunt end

techniques

using

cassettes

with

ends

This method nlso uses ~1synchctic oligo casscttc‘as 3 bindi9lg rc+n for J second PCR primer. It differs, howcvrr, in scvrral rcspccts fiunl the previous tcchni4uc.s. Tlx main ditfcr-ncc is that the locus-spccitic prinlcr 1;I is first cxtct ~tlcd by the DNA pdyrnerasc (scqt~ctlilsc) to the c.-9d of the fragnlcnt of interest, bcforc the cxscttc is ligated to the gcncrxcd blunt end is th91 r:irrid out using (Fifi. .3). 1’Cfi :1lllpliticiltio91 .I secorli~ 9le.ecxi locu+spccitic priiiicr I b 3nd ;I casscetc primer 2 h) iowcd by ;mothcr ;iniplitication with 3 ft9rehrr nc’sTSxi prinicr I c and prinlcr 3 The use ofscvcral nested rri919crs in this 9ncthod is ncccssxy in order to obtain ;I I,ib,h drgrcc of specitkity. Although hluntend li@on izSnot very r&blc and the tncthod SCCIIIC to bc q”itc t .dious, it hns been used succcssf&y for nnnlysi99g fln99iting scc~n~19ccs in pcnomic DNA tro9n coinplcx eukavotic orgnisn61”-‘r.

Lilllll

_____._____.__.__.__---.--.*-~

I

0 Amplification blunt

“r;;

PCR with primers lband2 1 __.__.~_.._.~*._~~__*._.= +-G

1

biotin

---

-~-l-------____.I.--.I_-

Figure 3 Amplification kechniques USIII~%&end

a/onX

casseltest”i8.

fktors. For cxnnplr, the h99nxr99 gcnimlc 19~9s;9 six of ,?Xl(i”’ . I-iwirirti ~c110n1ic IINA initially digercd with n rir:riction i’~‘yrnr ntight
chrornosomal

LXX4

by

capture

WC, have rccclitly

1 ._--. ..-,

walking

??PCR

PCR with primers lcand2

Froposcd

;i ditE:rctit

nmplif&ion

strdfcby which t;ks advan!syc of the powcrfiil biotin/strcptavidin sy:.tci ii to cdpturc hiotiiiylatrd fr,igc*.
;nnpliticatio*~r nxceion rising ;I specific biotinybecd primer ta t: xx by introducing biotin 3s 3 scpxrntion I&cl into tl-s specific restriction fmgnicnt containii>g pare of tlx known locus ;iiid the tlankiri~ rcgitru (Fig. 4). TI:c s;xcific biotinylat~:d KX fragrncllts ilrC tllcn iso,;-ted f&l the comp:r::g gcnoniic n9ixturc usi99g magxtic bends coaecd w,th strcptaviciin, and pl9rifkd by scvcc11 wnshing steps. The isolatioii seep is vc9y iinporkmt to cnxirc hi& spcciticity ofthc walking procrdurc, bccausc it rcduccs the conlplexity ofthc misturc by scvcr:rl ordcn ofnx9gnit~9dc. This is the key to r&blc walking in conlplcu gcllorllcs. 7’lw ;mchorcd sin&stranded tcniplatc is then csponcntially amplified using the locus-spscific prinxx 13 (but without bioein) 2nd 3 casccttr-specific prirncr 2, 111 order to product large nnlo99nes of chc dcsircd fiagnicnt for fi9rthcr :In;ilysis, ,I r~-.9l~rplifis;ieior~ step is cilrricd ollt. ‘fllc IIsc’0f.l licstcd locns-specific prinrcr I b ciisnrcs tli;it only thL* ticsired fragn~ctit will bc hrthr :u~iplifid Tlhs step is ncccss~ry bct:;iuec the

biotinylaeed pritncr 1 ;L niifihi an~wal scvcrd tinlca within a g&n gnornc (reasons for this include: the printer is not 3 sir&-copy scqusncc, nnncding conditions arc not strin~cnt enough, end the lnrgc ~~901nc six) ;9nd soinc nonspecific cstcitsion product9 could

41

cloning techniqw known locus

br fonncd during t!lc linear nlnplificxioI1 reaction, in 2dditic.n to thr dcsircd fmginci-.t. Sine all thcsc cxtcnsion products I :~ri-vt.:C biotin I&cl, they WC Ci&ptllWd on the strcpt;ividin bei& aid c:lrricd thfough the nicthod. Fin;ilIy, 1%X products an lx scqucnccd easily fironi both cntls without nny subcloning. This tnctboci is very rcii;~bic and &ws scyucntial using gcnomic DNA front complex walking eukaryotic organisms (yeast, nematode, hulnan) 1-‘J’. In pnrticular, WC have cnlpioyed the method for the following applicntions: ?? isolating of tlnnking regions in co~npics cukaryotic

genomic DNA A

I

linear PCR using one biotin-labelled losses-specific primer

I

along clmmoso~mlDNA fronl n givL\n ,t:lrting p;,int ior scvcr;\l kilo!l;lscs; 6 identifying cson-intron bonndarics within grim st:jrtitig frcbnr cl)NA scyucncc infornlation; * idcntifjling breakpoints (d&ions, insertions) in individuals without any cloning: * cxtcnding nlicroclisscctioll clones”‘; ?? IdcntitjGng and scqucncing unclonabic loci; 0 identifying YAC tcnnini for physic;11 11xlppi11g (C. 1'.Jmcs andA. IXoscnthnl, ul~puhlis)~:~d); of incr~nq~icte cl”WJA ~~~olcculcs ??clong&>n (II S. C. Jonesad A. Koscnthal, unpubiishcd). atid future

trends

;I ccrtLl

dcgrce

of dcgcncratcd

isolation of polymer-bound blot&-lahelLd products using atreptavidin-coated magnetic beads 2

/

1 r

ii;iF

I

I- Biotin&reptavidin-

*-lb -

la f

s+onerttial amplification of the polymer.bound singiu-stranded DNS, template using primers la and 2

I

re-ampliiication of an aliquot of the PCR product from step above using primers 1 b and 2 1

I

m

(

t

All IXX-based nicthods for ampli$ing unkltow~~ 3djaccnt to known loci LI:.C‘ rcstrictiolr scqu~llces cnzynm to gcncratc frqgnicnts prior to cithcr circularization or lighm of oligo c;Isscttcs to their ends. It is csscnti;~I in nil these methods that .tt lcast one rcstriction fragncnt IS produced which contains par: of the known locus. uvd is not toc~ kg:” to bc cfficicntly anlplifL+ i/j IMP. Since the distribution of the I r:striction c11zy1,~~ ,Iis usually u11k110w11 bcforc: starting the walking zspcrinicnt, a combination of scvcral ditErcst zlnzyrncs h;n to bc used in order to incrcasr the prob;Mity of g7icrating such fragnlcnts. Another appro;cch is co USC partial digestions of gcnomic I)NA with 3 colnbinntion of two ‘frcqucnt cnzynlcs. After sizing the DNA on ;grosc, the cuttd &ctions between 0.5 .~nd S kb :W recovered. The DNA can then bc used in combination with the ;mplitication sclx~cs discuspcasiLfc ifn rqiml of the gcnomc is totally resistant to rcstrictioir-ctizyttIrl dig&on, or highly rcpctitivc so that the nvaii~Ms sqwixc inforniation nl:ly pi’cvc:nt the design of one or twc’ iocusspccitir pritncrs, Thcrr is iI need for nlorc universal PCX :valking sci~cwws which do not rrly 011 m initial n~nnipui:~tion of genornic DNA (such IS &~qqz with restriction cnzyrncs and ligation). 011~ way to address this problcnl is to LISC a combination of a locus-specific ptlmcr and;I set ofrandom walking prinxx+ or sh*.rt pri~ncrs with

Biotin Biotin Eiotin

4

* 4

?? walking

limitations

1

-

gCIlOlW2S;

Current

a

hasa

Direct sequencing of the PCR product Figure 4 PCRwalkingalong chtomosonialDNA by biotinca&eW

1

2

3

4 s 6 7 8 9

in the TIBTECH JAN/FEE 1992 iVOL 101

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