Bacterial Cloning

Systems
I AN DUNHAM, KEN DEWAR,
UNG-JI N KI M, AND MARK T. ROSS
The field of
geome ana
ysis has recently seen a resurgence n the developreni of genom c
DNA I brar e; usl ng E col l host s. f he ease of appl i cat i on, coni ng capaci v, ad re
j abi l i t y
oi
baclerial cloning ststems make them
jdea
foT most genorne ana ysis experlmenls, ncludirg
large sca e
physjcal mapping and
genoric sequencing. The use of these liblrjes as comron
reurces has been facilitateci by the developrent of
gridded ibraries and a seres of high
t hroughput
prot ocos f or hancl l ng ancl anal yzl ng arge numbe| s of cl ones Thi s chapt er
clescribes the general cons deraLions and
procedures in the consiruct o, repllcation, storage,
ad ana ysi s oi bact eri al l i brar es. The t echnl ques and examp es d scussed are based
pri mar'
i l y on t he use of cosmi d, f osri cJ, P1, BAC, and PAC c ones and l brri es f or genore anal ys s'
For deta led Drotocols on library conskuction and clone manlpulation of the cosm d syslem,
see Evans et al.
(this
vo ume); for bacter ophage P1 vectors, see Sternberg
(lh
s volume); nd
for BACS, see Biren et al.
(thls
volurne).
Chpter I Bacterial Clorng Svstems
Genomic Libraries
During 1he I980s, cloning vuct.rrs such as plasmids'
baccriophage l, or cosmids were used lor ihc con-
strlrclion ol genonic DNA libraries Wilh t11e
exceplion ol systematic
pproaches !o ihe analysis
of sonl e smal l genomes (coul son et al - 1986; ol son
e! al. 198; I(ohara et al. 1987), mosl irvestigators
aimed at isolating a genomic lragmenl conla rng
the gene or gene clr.rsler ol intercst. Tbe small inserl
size-of these types ol clones and the linliled siTe aDd
r1 mber ol the regions undcr invcsligation
favorecl
the isolation ol cloncs by screeni
g replicas ol ran_
doml ) pl dr co l i br J e\ '
(
n' r r L
' r r r ' b a' r d s eet
' r g
these Iibraries was r1ot dilficull, being accomplished
r l r uuel r
pr ocedur es l r e" d) i n r r ' - i r n- o' r n' ol ( ( u
l " i ! i ; 1. ' ; y l bo, , . | | cr I n ddi r i ' r ' l r c ' u' r r r s
efficiency for these sYslems was high, thus, a single
jnvesiigator
corid rapidly creatc new libraries with
small amonis ol DNA Hence, with thc focus otr
olating a specific region ol iDter(st, thcre was lrt
tle need !o maintain rhe libraries as permancnt
The abiljry ro clore megabase DNA lragnents
as YACS (Iluke et a]. 1987) fundamenlally
changecl
genome mappig and analysis
(see Green et al ,
lhis volume). But as valuable as YACS proved to lrc,
YAC library costruction was dilfi.ult aDd labori_
ous. The poduclion ol YAC libraries lirst involvcd
masteriDg a whole series ol new techniques Alter
these techniques were learnd, a prolonged period
of "proluction" work ensued, in which a rem ol
investigaors would
perforrn he same
proceLlues
reDeatedly Io accumulaLe sullicient numbers of
cl;nes to seneraie
a librarl'. Th s, cach clore iu a
YAC library represenled a precious resorce that
col d be recrcaled in only a lew iaboralories a 'l
even rhel] would require a serioris inveslmerl ot
time and labor. When a library is arayecl. ' e,
when each oi lhe original tanslomants individ-
ually picked into a single well ol a multiwell
plaie,
it becomes more easily repli.aled, stored, scrcereci,
add shared. In this wa, clones and libraries at
would have otherwise been precious or rare can be
readily shared and utilized by those outside of lhe
laboratory in which rhe librry was
Scnerred
The
abiiiy to distribute copics of lhc librades, and lhe
filers ard DNA pools needed o screen thclrl, has
been a major conlribtor !o our abiliY to obtain
and study genomic cloncs llecause the 'lons 'n
anayed libraries have permanent addresses, thy
can be recovercd repcaedly over a perjod ol year!
l r o' r ' br . ' Jr ' l l r r oJj ho r l l l r e r ( ' c" r ' 1" ^ nn
r r r i
ty. In addj!ion, ltre addresses of arraved clones ca':
cisilv be sharcd among rvestigalors who hv'
occeis to tbe safle library along with ilrfonnalior:
about the sequenccs lhey contaiu l this way cla
abc't specific cloncs car acclrmulare ard the clon\
becorne even more valuable ihrough the 'ontrLblr
: un
^ l sn- \ r r \ i n , l I Fr . nl I hu dr or r c
' n
J \ v" \
n.
! . . i t , , e
r a. r \ r ' ur r " r r d) ( , 1
gi nor ' ' . l r
' - f i '
i he advent of l arge-scal e
gerome anal vsi s hai
been accompanicd
by lwo major changcs ]n ln
way bacrei al cl ones al rd l i brri es are uscd' Fi nr
techical advanccs havc produced a new gnera
Li on ol vcLors l hal are capabl e ol cl oni rrg
gen(t ur'
regi o$ ol up l o 100 kb Thes l arge' rsen ' l onl ne
veci ors, i ncl udi ng Pl (S! errberg 1990), BAL
(Shi zuya et al . r992), and PAC (l oannou er l
t oq4, . dl t os r J p. ddn. r ' y ' i . . 1
r Fgr r ' . ' pdnr r i r
g- \
eral megabases. Thi s provi des Lhe mcans 1o sl ud\
even l arge (e. g. , n1al n al i an) gcnomes 1 ' l eL!
usi ng bacl eri al cLoDes. second, t hc need l o anal yzc
l arge regi ons sysl emal l cal l y has spurred t be deYcL
ot mr nr
^ l gr ' dd. . l ' l ' f " r ' c
^ l l cr r np J( ' l ; ' r ' r r r i
coverage
(i . e. , >l 0x) l or n nl Der c)1 orgi snl s
The resurgence in the use of bacrerial clonin!
svstems has been due to the development ol rlensc
ly popuLatecl STS physical maps (botb fro YAC
based and radiation-hybrid
mapprg ICollins
cr ai
1995; Hul i son el 1. 1995i Schul cr et al l 997l ) anJ
the increased capacity for cloning large
jrrserr
DN1
f ragl l l enl s. Al l hough t he. l oni ng capaci t y of Pl t
PAas. and BACS is lafge corpared !o rhal ol 'oi
rnids, it does rot approach ihat ol YACs H()wever
thcre are several advamrages 1o tbe use ol bacteri
(loning sysems over YACs. BacteiaL clones in los
copy numbcr vecl ors, e g, BAC, PAC, and t ()sl rkr
are hi ghl y sLabl e ancl sbow reduced ' l rmref r! n
compared 1o YACS l n addi l i o' bacl eri l ' el l cu! '
ures are easi er t o DraDi pul al e t han yeaf cul t Ll rt r
bact eri al cl ones aDd l i brari cs grow norc raprl i
than YACS, are casier io grid and handle, and gro$
to higher celL dcnsiy, which allows ior nrore cIIi
ci enl scrceni ng. Mof i mponant l f reasonabl y hi gl
l uart i l i es ol i n! act ,
pure DNA l ronr bact eri al cl on('
can be ol al ed wi l houi l he copu. i f i cai i on ol ! . :
genomi c DNA, r. rsi ng si rnpl e pl asl i d ba\ ed DN:
preparaLions.
Many investigators will ned !o coDslruct net
genomi c l i brari cs l or hci r. l oni ng and mapprn:
stLrdies. Howevct a growing rlrmber of iibraries atu
readily available i the loml of lrozeD plales t'-
arrayed cl ones, i i hers f or scrccni ng by l rybri di za-
| , n , r
LrNA pour.
u D,
P-h ed . . rperrr g Tl
p
" \
r i l " l
j ) ' r r ' r
l r .
, \ r
' \ .
" , P
n i nv I b d
rori es f rorn spendi ng l argc n] ol rnl s of l i me and
rcsources recreat i ng l i brari es l h1 af e sl ready sui t ed
1() t hi r needs. t hereby al l oi vi ng smal l -scal e cLoni ng
and mappi ng el f o s t o rakc use of t he mof
advanccd
(l ooi ng 1chni ques. Cenonl i c l i bra. i cs
ol f eri ng dep covcrgc l rom a vari el y ot sp: ' s arc
now acccssi bl c Lhf oLrgh ccnt rl l ci l i l i cs or cornp-
nies. The world wi(lc wb addresses lor som ol
t hes l i brary soLrr(ci are l i st ed bel ow.
Calilonia lnfitutc of Tc.hnokigy
ht t p: / / ! t u, Lr e. . aLt ec h. edu
Cl cI nson Uni ver si l y, Ger ome Cr t ( - ' f
ht i
p:
/ / genone. cl ems. r . . : ( l u/ addex. ht r 1
Roswel l Par k Cancer I nsl i t Lr t c
ht t p: / / bi c p c - r nd. buI f a l o. edu
Genonc Sysl f l ns I r 1c.
ht t P: / / r . d - qedomesYst ems. con/
Re5ear ch Cer t i cs
ht t p: / / l v, r c: j qcn. co r /
German Hrnran Gcnorne Project: I{ZPD
ht Lr r : / / ! / w! . r zpd. de/
Construction of Bacterial
Genomi c Li brari es
Importnt fclors to bc considerd whcn colrstrcl-
i ng or rvorki Dg wi l h l i brari es f or genome wi de
aoal ysi s arc summari zd bel ow. For det ai l ed prol o
cols on librry confrr.rction using the cosnid sys
rem, see Evns ct 1. (rhi s vol ume); l or bact eri o-
l hage
Pi vct ors, scc Srernberg (t hi s vol umc); and
I or BACS, see Bi rren ct 1.
l t hi s
vol unre).
CHARAC ERI STI CS OF DI FFERENT BACTERI AL
CLONI NG SYSTEI I I S
The divcrsily ol bacteial clog systems available
f or use i n vaoLrs st ri ns of . . ol i enabl cs i nvcsl i -
glors to choosr thc chning system bcst suitcd lol
stecilic eperinrcnls. Table I summarizcs vaiety
, ' v , . r or . r . Fd , r ' J r l r I r , r . t er , l l or i r g. ) .
rems and d(scri bes t he l eat urs ol t hese 1' ct ors
rommonl y sed i n genome anal ysi s.
Constructon of Bcterial Genomic Lbies
Unt i l t he devel opment ol t hc ncwcr cl oni i t g
sysl crns, cosni ds were wi dcl y uscd. f or cl oni ng
largc-insert DN,^. hagments in . c01i. Cosmi.ls have
lhe advlge of the high efficienq, oi rr-rcdi-
aed bacl cri ophage , packagi ng. ht addi t i on, cos-
ni ds havc t he advart : j es ol a sml l vcct or (c. g. , a
pl.snlid) bccause ol recir{ulariztion ci lhc DNA to
a plasmid forr rirhin lhe host cell. Cosmids allow
rhe cl oni ng of l 5
-45
kb ol genomi c DN and have
beru sed t o bui l d physi cl rrps corposcd ol
overlappirg cloncs in hLrmn genomes and those o{
orher organisms. Cosnlids exist as multiple copies
per cell, wtrich llows recovry o{ large amounrs of
DNA duri ng DN, { prparal i o l rut can l ed 1o
rearrangernenl s of t he rl rd DNA (Ki m cl a] .
1992
).
Procedures f or t he use of corni ds i n genome
anal ysi s af e descri bed i n Evans (t t ri s vol ume) (see
al so Evans c1 al . 1992). The l osri d vcct o (Ki m et
al . 1992) conl ai t l s ccs si t es f or cor ncdi at cd bc1e-
ri opl )agc )" packagi rg, bul I i kc BACS, i l i s deri ved
I J r r " r . ' i r gl e. oo\ | l d' r u' u / . o/ . . \ L' I e
fosmid vector llows construction ol libraries bear-
irlg cosrnid sized insc]s that are rrore stably main
taied during propagalion than those of convn
The insc.t size in cosDrid and iosnid clons is
l i mi l cd by t he consl rai nrs of
f ckagi ng
t he DNA
i nt o rhc bct eri ophage l ,
l hagc
hcad. The Pl
cl oni rg syl cm (St emberg 1990; Pi crcc cr al . 1992)
packages (Loned DNA nr the larg-r bactcriophage
Pl phage head, al l owi ng cl oned f ragnent s t o be as
I arge as 100 kt r (see St e berg, t hi s vohl mc). The Pl
vect or i s si ngl e-copy vect ot ol l cri ng t he advan'
rage ol inscrt srability. Small modifications to tbe Pl
vector. and a diflerent mhod ol delivering reco
binant moleculcs inlo the host t. coli cells (cleclro-
lorarion
inread lrl trarrfection), 1ed io thc devel'
opnent of the PAC sysiem (IoaDnou et al. 1994)
aDd the clonirg o[ DNA lragments hundrcds ol
ki l obascs i n si ze. Si mi l arl y, rhe BAC syst enl
(Shi zuya et al . 1992), whi ch uses an F l act or-
(icivcd vector has becn used to corfruct librrics
of st at rl y propagat d DNA l rgment s as l arge as 300
kb.
AVERAGE I NSERT SI ZE AND REPRESENTATI ON
OF THE GENOI V] E
In many gcnomic cloning stralcgies, DNA so rces
and preparations can b ma]]ipdated to cnri.h lor
speciiic rcgions or targcls ol interest- Howevet for
g
E
: 9
l L^l
I IF

I
I l E
-
9. 1
L l : r=l ; I i : =l
I
E ?n- 5
: r i E
: l : P, c - : v , j l
L El ; : ; ":
;
j t \
i
I _l | :
l - , ' -
o. L-
l ! . l l ; ' j P
- { ' - l
:
i t
-
^
: , . t 7
l z
/ "
| : ,
l -,"1 l l :
l i 1. ; v ; L
l L
l :
": l
i
"+
: t | : a'
i ;
l i {l q
- ": ,
==
1= l . l , o
! . l
' -
-
t :
"
t - L t F8
Lq I E:
]:i l ' ! e e ; : rl !:i
t e: : : : i i
6i
: : j
lErlE
q:
; H: Hq H; i+1
t . . :
. r : . i
, i ! , . qst =- : ,
f i ' .
I : ; I
i
=
F ;
'
-r
'
.
'
-
'
i F
]
'
i
,
:
l t-l l ;' :"
| i 4 E I
sl l
-
l ! , - -
" ^
" : : i
l l l L
r* I ! !;1.!i i
I
tl
l rt.
I
a
^.
. -
= - . i . ; : "
ili rl
:
:
iil.;:
'
l Lt
.-{ "- :
i " l ,bE,
rl
rl . t:

f
-?l !
;e
i l
-
, " - : : !
. : I l , ; 3
- . ^l
: l i a" -
! *.1.
",
; i e FP:::
. ql i i
- -
i r : e. . 1 1; !
J c
l o
o-
"
. ! " s
rtF
I l1;:
:
-
| ; i . : - .
: l l
q
- l Fr ' :
il ll; -; : : l:.,"
3l !l ;
=i - : .' l "' ";":
Chapter 1 r Bcterial Ctoning Systems
Construction of Bacter;al Gercmc Libra es
libraries intended for genome'wide studies. the
, \ pf ul nF. . ^ f
I r e
l i br r y der " . r , l .
^ r
n\ i mr / i r . E
rhe proporl i on ol i he enl i re genore prsent i n t he
librry. Fo a gcnorrc ol a givcn size, tbis is deter-
mind by two characteristics: (l) the
jnsefi
size ol
cl oncs
j n
i he l i brary and (2) t h numbe ol cl ones
' I
r l . l f . J r y t r r . r . \ ^ nr ' i r i r - .
1, , . , . g. r ' ni ,
libraries are usually charactedzed by th size ot thc
library. i.e., ihe nLnnber of clones within the library
t rd l l l c ncn i nscrl si Te ol t hese cl ones.
I nsert si ze i s of cruci al i nport ance i n gcnone
anl ysi s bccuse i l conl ri bures 1(l t he overal l
genome coverage of rhe Library (se below) and is a
cri l i cal paramel er t o t he overal l success of t he pro-
ccdurcs i n
l t enomc
anal ysi s. I l t he goal i s t o spaD a
large regil where markers are available al a mean
sfacing of 1 per 100 kb, clores with a mean insert
siz of 50 kb will rarly link two markefs, nd an
al emat i ve met hod f or obt ahi ng overl appi ng
clmes, such as lingerprinring or walking, will be
" qui r ed.
O r l l r e ur her h, nd i f r l , c r n. dr i n\ ,
-
\ i / e
of the lilriary is 150 kb, scrccning wilh rnarkers at
d n F" r t , , i ns of I per l 00l bdl q! \ umc c Sons
Io be isolated as contiguous ovrlapping clones.
sinlilarly, ihe isolation of a gnomic region from a
library with large inserts is accomplished by scrccn-
ing, isolaling, and analyziDg lewer clones, simplify-
ing the genefal togisril:s o{ ihe projcct- Larger
inserts may be impofiant to cover regions with
nusual sqLi ences cont ai ni ng a l ow f requercy ol
restrictior sites. Howcvcr, thcre my be cases
Nhcrc incrcased insert size represents an impcdi-
ment , e. g. , t he assembl y ol a andom rhot gun
sequence ol a clone several hrudrds ol kilobases
ir sizc coLrld present problems lor the astbly
Th size of the library is similarly important.
Thc goal ot lib.ary confruction is lo maxlmize the
1i kl j hood of l i di ng t l easl one cl one, and usual l y
mul t i pl e cl ones, I o a y I ocs oi i nt eresi . The prob-
bi l i t y (P) of i sol aLi ng cl ones f or any regi on oi t he
ee ore increases as the library size increascs (see
elow). Furlherrnore, in genome analysis, a major
Soi l
of screci ng l i brari es i s t o i sol ai a seri cs ol
. orl i gous overl appi ng cl ones spanni ng l arge
.egions. Iusl as the n nber of clones that cn be
rsolated repcscnling a single locus ircreases wjlh
:he library size, lirc lcngth ot lhe continuous pattr
rde up of overlapping clones that can be general-
cd i ncrcascs wi t h l i brary si ze-
Characterizirg clores with espcct to ovelap is
:rsed ro (1) orient clones with respect to cach olhet
'lr
allow the determination of paths of clones
across a rcgion, and (l) provde evidence ol clone
lidelity to the
lenome.
The extent ol ovcrlap
'between
clores in a library increases as the size o{
lhe library increases. Altho gh there are advaD-
t ges t o usi ng l arge l i brari es i n genome anal ysi s
proiects, there ar also practical and economic lim-
its to their production, storage. ard manipulatioD
(see bel ow).
Consi deri rg t hc i ssucs di scnssed ai l ove, hol v
l arge docs i i brary necd t o bc? Thc 10! al amount ol
genomi c DNA present i n t he popul at i on of cl ones
i n a l i brary i s urrl l y severl t i mes hi gher t han t he
mount ol DNA i n t he genome of i nt rest . The
ra(i o of t he amount ol genomi c DNA i n t he l i brary
to thc ariounl to DNA in thc gcnome is relered to
as the nu ber o{ genome equivalents, the depth ol
genome coverage, or he genome edundancy. This
is ofren abbreviated as lx or "l hit," *'hich rclefs lo
one genome equivalent. The calculatlon of a
library's genome coveag involves lour lactorsr (l
)
the genone size ofthe organjsm, (2) ihe total nm-
ber oI cloncs in thc library, (l) lhe nmber ol llon-
contributing cloDes (i.e., empry wclls, vcclor only
clones) with the library and (4) thc averagc
insrr size of e cortdbuing cloncs. Thc lormula
for determiring gerome coverage (c) is c
= (total
number of clones
-
noncontributing clones) x aver'
age inseft sizelhaploid genoine sizc.
The coverage value indicates, on avcfage, how
many clones containing a parlicular locus will bc
pescnl. However, a genorn coveraSe value greater
rhan I does not meaD that the cntirc gcnome is
presenl
jn
lhc library Instead, certain regiors wiI
be present less olien (or cven absenl) and other
regions wil be presenr nore oltcn than the average
umber suggest ed by t he coveage val uc.
^ssuming a random cloning of geDomic DNA irag
ment s, t he probabi l i l y ot havi ng part i cul ar
genomic locus present in lltc library {ollows a
Poisson disrriburion. Tablc 2 shows the probabilities
of findiDg a locs wjilin a library Io libraries with
genome covcagcs iom 0.5 1o 10, assuming at
. thegnomc sizc is rnore lhan lo0 times large than
These probabities (scc Table 2) are an overes
timatior duc to e assrrm ion of random clorlillg,
i.e., all of the genomic rcglons ae equally clonable.
ln reality, some regions are easily cloncd, wheeas
other regions are dilliculr or impossible to .lone.
Although thee are numerous lactors that con-
tribule to the nonfandom bias ol libraies loward
cefain gcnomic regions, two are mosl noable.
First, the restriction enzyme rcogniiion sites tnat
Chapter 1 r Bacterial Cloning Syslems
Table 2 Probabiliq ol Having One or More Clones/
LocLrs within a Library as a Functior ol Library Size
f bl e 3 \ J ' 1b. ' al on( \ ReqJ i . d l or 7 1>
Genomic coverage
no. ol no. of 384 weu
Eaploid
( Mb) ( kb)
0. t
t l
2
)
4
5
7
9
l 0
20
1000
1000
1000
50
t 00
50
150
a
20
t 172
l 9l
l 9. l
63.2
86. 4
95. 0
98. 2
99. i
99. 75
99. 91
99. 99
99. 995
1000
7504
450, 000
150, 000
Th probabiliry ol lindnrg clones Ion litrrv ol. Loverage
js
P(r) =
= .-
TIre
trobabihies
shown ae ihos. ollidig oe o m' re rlnes
Ir
P(o)l Jo librrries wilL gclome.olcmges I.om 0 t lo lo
are used for cloning are rot unifolrlrly distribLrte.i
in geDomic DNA. Second, rhere ae regions within
genumr c \ equen\ c\ r l r " . d
( cl hl or pur l ) r ' ' di n
tained in bacteial host cells lor these reasons, ]t ts
usually necessary !o conslruct libraries lo a greater
dep, n l l ' r r . , r gB<ned L' v. r ' e Poi ' 5un
f t r l i ,
i ^ n
The depth oi genome coverage required for a
genomic libtary depends on tbe purpose of the
library. As seen in Table 2, it is very likely rh1 a 5x
library would be adcquate for linding one of more
clones for a locus of inleref, since more than 99yo
ot lhe genome shor d be present.ln a proje{r where
only a few clones per locLrs are needed, lhe addi'
tional reagenls and eflor! required io cosrrrr't a
l " r g. l 0' , . r " - " r v ar L , . ^ $or r r nl ed Ho{ e\ r r ' I
walking or mapping, where each clone h a sees ol
clones must be identificd, he chance of fiiding no
clones at some point in the parh is grealer (being rhe
product
()1
the probabilities ol finding eac! ol e
clores). Ths, on a shtistical basis, e g., when con'
slructing clone contigs lrom a 5x BAC library, gaps
are expected, on average, every l0 Mb. Librades ot
greater depth (>l0x) not oniY provide an in'reased
. i kel ' l r ood ul hui l d' ng
( " r ' i nr ' u. r \ ! ovcr cac ov(
long paths, bul also allow a flexibility in selecting
specific paths (e.g., a path of minimaUy overlapping
clones tor sequencing). Thus, lhe appropriate num-
ber of clones in a librarY should be the miniral
number ol clones giving a genome coverage likcly
to meel the necessary reqldrements
CoDstrucling and screeing highly redundarl
libraries for large genornes requires generating and
handling many clones. This increases the cost and
elfort for dreir production, storage, and manipula_
lion. Table I sumlnarizes the nutbers of clor,es
required to prod ce 7.5x coverage libraries for
organisms of dilferent
geiome sizes Libraries tor
organisms with smaller
genomes requjre smll
numbers of clones. Fo inslance, a 7 5x fosm;d
library for an organism with a 20 Mb genome
cor d be slored in a set ol cight 184-well mjcrotier
pLaes. A 7. 5x human (georne si ze of 1000 Mbl
BAC l i brary wi rh an avcrage i nscn si ze of 150 kb
would occupy hndreds ol mjcrodter plales; a 7'5x
h man cosmid library woukt occupy more than
1000 pl at es.
DNA SOURCE
A genomic library relleclslhe genome holn which iI
was made. The selection of parlicular individuals.
srains, or cell lines as a DNA source can cnhnce
charactcristics of Lhe resullnrg braries For instance
io cloe Lhe sequerces adiacnt to a specific trando
cation breakpoint, one iglt choose ro constru( Ihe
library lrom ihe DNA of lhe indlvidual wilh that
breakpoini. Howevet in c context oI geDome
analysis, he DNA source is most olten chosen lor ill
representati('r of the "normai" configuratior of the
genome, rathcr lhan for the presence or absence of
specilic attributes. Even ilr lhe laiter case, imporlanl
issues rnusr be considered, e.g., the use ot tlaJls
lbrmed cell lines. If a cell line has beeD exensivelv
pu ' " 8ed. i r i \ l r \ el v . l , dt c
" .
Jn, ul r ed mr r r i n.
and rearrangemens that deviate fuom lhe
"nomal-
stare ol that genome, and lhus an early passage cell
line would be a betler choice The sex of rhe orga
ism that will sewe as the DNA source may also be a
factor. In malnmals, twice as many cloncs must be
i
s.re(ncd tor X chrornosone loci whcn DNA tron a
malc is used. compared to rvhen DNA lrom a iemlc
is used. Y chromosome loci will nol be preseni in
DN^ from fenrales. If it is likely at clones may be
used larer ir hrnctionl knockout or honologous
reconbinaion experiments. there ay be advar
tags ro using DNA derived from the same animal
rrain as the recipienl ccll. For the human genome,
tLrral variation becomes signilicant. To obtain a
library that minimizes vaiation, DNA lrorn a con_
sangLri neous i ndi vi dual coul d be used; f or a spcci l i c
clllomoso , the chromosome could be flow-sorled
Irom a monoclrromosomal somaiic ccll hybrid on
(hc
olhe hand, to maxinri?e the varialions l}lai carr
b identilicd lrom the clones in lhe libfary, DNA
Iol multiple individuals could be pooled. Finally,
erhical aDd legal issues nlust also be considered. For
large scal humn DNA sequenci\g, guideles lrave
hccn issued lor rec.i|ment and proteclion oi DNA
donors For iformation on these guidelines, sec the
^"alional Ccrle for Hulllan Genome Rescarch/
Dcpalment ol Energy (NClGR-DOE) Guidance on
Hunan Subj ect s I ssucs i n Large Scal c DNA
ScqLrcnci ng at i he f ol l owi ng Worl d Wi de Web
ht t p: / / \ \ . ornL.
qowe/ Tect i Resou. ces /
H'nan Genone/arctrive/nchgrdoe hLml
AI \ OUNT AND QUALI TY OF DNA REQUI RED
The quantity ol DNA needed to constr cl a library
f . i l l vay depedi ng on t h cl oni ng syst em chosen
rnd e ext ent oi genome covcragc needed (see
Tblc 4). As library consluction ofien inl,olves sv_
r:l rounds ol DNA preparation, size-selection, lig
iion. and lranslollnalion, wilh optnization oi
Construction of Bacterial Genomic Lbraries t
each fep, it is important to be able to oblain large
amounts oi DNA initially or to be able 1() repeat the
DN^ preparalion as needed. Diifcrcnt bacterial
cloning vectors can accept different size rarges ol
geomic inserr DNA| fiom 15
-45
kb for fosmid/
cosnids ro as much as 300 kb {or BACS/PACS. This
has irnpoftanr implications for the preparation ol
the source DNA. In geneal, the average size oI
hagments lrom the p dlied souce DNA mrsi be
five to six timcs larger tiran the size ol lragments
equired in the ligation slcp. In particula, thc
preparation oi DNA for the construction ol B^C
and PAC libraries uses source DNAS isolated in
agarose pl ugs (Shi zuya et al . 1992; l oanno et a] .
1994; I <i m et al . 1996; I oannou and de Jong 1996)
and eriails gende manipulalion of the DNA and
PFG! to obtain suitable material for cloning. The
preparation of source IINA lom cells enrbcdded in
agarose permits the isolation o{ very largc DNA
mol ecul es, whi ch f t er part i al di Sest i on wi t h
reslriclion enzymes and size selection on PFGS
yields genomic DNA fuagnenls lhat are hundreds
oL kilobases in size (see Rietllma ct 1. 1997, Vo].
l). DNA prepaed in aqueous solution is more sus
ceplible to shcar-indLIced breakage, llhough it car
be of suflicient lenglh lo be suitable lor cloning in&)
cosmid and losmid vectors.
THE BACTERIAL HOST STRAIN
Ceain DNA sequences may be dillicull to clone
and/or rnaintain in a bacterial host. Many E. rrli
murations have becrr identilied at improve ihe
abiljty o{ a strain to scrve as a host io ioreign DNA
T. r i I r r cf r i gl r l e' el " of r Ft p. i r i ! - \ . l uc r . e\ i n
lhe genomes ol higher organisms mears that mol-
ible copies of thcsc repetitive sequences wili be
rbre 4 clonins Merhods suiiablc ior Generalins Gnomic Libraries u.i"sl!{l!!9llg{4
l l i f e enr
f
r ef r at ' ons
(pg) " Approfjte cloning syster for sourcc DNA
arn(Di. DNA in agarose
:-nomic DN^ nr aqlrcous
-:.n rmic DN^ in queous
a: r ] r r l ed chr omoso r a!
l\.\
: - : : . : i ol ar ed Y^C DNA
BAC, PAC, Pl . { osmi d, cosmi d
rl, losrid, cornid, bacLerjophagc ,, Ml l,
flasmid
bacreriophage ., Mll, plsnrids
losnrid, cosmid, baclerlophagc ., Mt:1, plaeids
fosmid, cosnid, baderiofhage ., M1l, plasmids
>l ML)
100 200 kb
50 100 kb
>200 kb
>100
>100
>100
0. 1- l . 0
0. 01- 0. t
Chp16r 1 | Bacterial Cloning systenrs
Dresent wltrin individal clones' Wild-tvpe
E crl
strains are highly
proficien! in recombiDalion'
whi ch causes del e! i ons
ad rearrangemenl s
through ]lramolecrlar
ecombilration
(and rn case
oL mutticopy veclors, inlermolecular
recomblna
tion as witl a lhese repeated sequences
Poor
cio,rabilitv ao resul$ lrom irrcopalibilily
oi lhe
Ioreien DNA with rhe natural host reslridion and
modiflcallon sysrems the choi.c of host strain is an
imDortal factor lor the elficieBcy oi lrane'lron
or
r.airstormaLlon,
and the
quantity ad
qualily oi
DNA obtaiel from cultures To rcduce the ability
ol !h bacterial host !o modily cloned DNA frag-
mcnts in such ways, scveral host strains wilh
mJr r r . eenol vpes
r e n" w r r l o' '
or l i br an
Lo' l \ r r r . : on L. ee
l bl . '
- '
l r r
Li cnr r l
DH5& n' j
l l Hl 0B are usel ul f or cosmi d l i brari es, and DHI oB
has beer used lor BAc and tosmid library con_
struclio.
Table 6 slrnmarizes
some ol rhe rvell
known E. rlt gencs that are directly or indiredlt
involved with DNA recombination
processes ano
host restricrion and modilicaiiotl,
or otherwis
inlhrence the sulability ol a slrain lo serve as a hor
lor clorcd liagments
Constuction of Bacterial Genomic Libaries
s
:
I
E
i
l , i
r i
H
2
ii* * *
i
- ! : t s s E
<
I
j
- :
il
3; i
=
F
355E
g
t;
i; ti e *
t"
!-
!i
;i
f: ;,!i i;
ii !! {: ie :i
i
i as ! i
<s
. : nE
, ! . ! F . : 3 Ed
j ^
: I ) -
aal ) zz
3
I

(,
3

10
Table
Chapler 1 Bacterial Cloning Systems
6 E. col Mulalions That Allect lhe Ability to Clone Marnmalian DNA in Dillerent Hof Strains
luncions and mutalioal effects of lhe genc
The major gcne involvd in homologous .ecou inatioo MlrralioD increascs the rabiliv oI DNA rhr would
ure.*i'" e p-"e rn delerions ot rcarransclenrs
(e'8
' DNA containing repcitive scqueDces
These gcnes encode exonuclease V and are also involved in ecomtrination Mutatio inoescs the rabilirv
oi DNAt hat woul . l o her wi sebe| r onet odel edonsor l car f angcmenl s( e' s. , DNAcont at nnr gr epct Ll t ve
The gee eD.o.tts a componc! ol the RccBC cDzvmc complcx and 's reccss'v tor e 'psio of rhe
cnzy;et nclcolylic adiviies MtatioD iticreases the stabiiitv ol DNA that would otherwise bc proDe ro
ilelerions or rcanieemenrs
(e.g, DNA conlaining repcdLive sc{luenccs)'
The gene enco.lcs componcnr ol dre ru' conbition
pahwav The r'' mutalD inproves rhe growln
The genc encodcs exoDuclease I, a coiponcL ol rbe ru'FPaLhwav The Jc nintario trprovcs tlre erown
ol rda aDd /e.c nlurams
Thc gene encodes a sul'uDit oI lhc esrictio enzvre Ec"( 'rdR uranls can methvlare DNA but are unable
lo estric! lorcig DNA a rbc apfropriarc recognition 5i1c
The gene encodes subunit oI E .ol; ierhvlse which mtllylales lhe a'leie rc:idsin,E(
rslricl'on
siles:Thc t (crricrion ezvDie lalls to recognize DNA clLvlaled al adenine residues DNA rr'm n J'll
r our an
. l r - r * r l
" e
r e l
. ' l ed
i - c
' - \ dt l ' o\ '
Thc gee eDcodes a
lrorei
subunit thai colers sile spe'ilicirv lo bo HstlM and HsdR
protcins /rri
rutan$ are in.apable oI both melhyladon and rcslridn
The gcne eDcodes a eslricrion enzyme. Muiation prevenis resliclion oI DNA ntthylaLed al Ihe scquedce
5--C;"CGG-3', thereby allowing morc elljcient.lonlng oI DNA conraining mclhvlcvrosie rcsjd"es
The n:ra and mdc
Senes
cocode the wo subunits ol the McrBC resricion endonucleasr' Mutation Dre
vcnls resrriction ai melbylcytosc ar lne scquencc 5-_GDcc_l- and thus allows rue cllicient cl ringolDNA
conaining 5 mcihylcyrosine or 5}vdroxvnreYl cvlosine'
The
Senc
encoites a refricrion enzylne Mu|alion
Prevorts
rerri'tion ol DNA tethylated a the sequences
:-_ci.46-r- n. 5--c-"Ac_l-. rhcreby atlowing mo.e ellicienr .lonilrg of DNA coirainiDg nrelhyladenne
The genc en.odcs en.lonuclese I DNA puitied lo'n 'J mltaDts is of a hjghe'
vleld
ad has rcreas'd
s!bilily due to thc absence oI is nuclcasc
The gene ecodes rhe rc!ressor ol thc /d opcron. A muttion in rhis genr aLlows kr irrc'caserl raDdonna
tion rlticieDcy ol large plasmids
lacz Ml5 This .lcletecl
sene
enco{les an inctive tom ol
p'galactosidase thaL lacks amino cids l l 4l Some clonins
vec or scncodeanami not cr ml nal f ' I f ag eDt oI pgal a. l osi dasc' Thi sal l o] vs( , compl e eDt al j onwi e
hosr / 4. ZAMl 5pr oduc! ol or manact i veenzyme. Thccl oni l r ssi l e( s) of suchvedol s] i eswi t hi nedf r ag.
ment ' e codnr gr egj onTher ef or e, no r ecombi nant sl or t nbl uecol oni csonmedi umco r ai ni ngxgal
whercas reconbinats are wbe
^^crA,
mdtsC, and mn all rcsrict DNA modilied by CpG ncrhvLase