3.2 Human foetal brain cDNA library construction
3.2.3 cDNA library arraying into microtitre plates
Initially 2 0 ,0 0 0 clones g enerated from the fraction #13 ligation w ere picked ro b o tically in co llabo ratio n w ith P eter Jo n es as d escrib ed for the m ouse adult brain cDNA library.
As part o f the d ev elo p m en t o f an integrated set o f autom ated p ro ced u res for the h ighly parallel an aly sis o f m any th o u sa n d s o f clones (M eier Ew ert et a l., 1993), a ro b o tic co lon y pick in g dev ice was d eveloped in the lab. The adv en t o f e x te n siv e co m p arativ e m apping p ro je cts, en c o m p assin g m ore than h alf a dozen d ifferen t o rg an ism s, and the in creased use o f array ed cDNA libraries have m ade the p ro ce ss o f p icking ran d o m ly plated colonies into m icrotitre plates a rate lim iting step. T here is a clear need fo r the autom ation o f this p ro c ess for w hich there w ere no com m ercial appliances at the time. A m achine was developed that is able to pick E. col i and y east colonies into m icrotitre plates at a rate o f 3 ,0 0 0 clones p er hour. A detailed d escrip tio n has been p u b lish ed in M aier et al. (1 9 94 a).
A fu rth e r 8 0 ,0 0 0 clones w ere arrayed into m icro titre plate analogs, u sin g the ro b o tic colony pick in g m achine d eveloped in the lab. A pproxim ately 95% o f w ells con tain ed g ro w n cu ltu re after o v e rn ig h t in cubation 37°C. T hese clones w ere picked into 38 4 -w ell plates (Q- plates) that have the sam e fo o tp rin t o f co n v en tio n al 9 6 -w ell m icrotitre p lates. The sp acin g b etw een w ell centres in 9 6 -w e ll plates is 9 mm, w h e re as that in Q -plates is 4 .5 mm. Q -plates th e re fo re contain tw ice as many ro w s (labelled A to P) and tw ice as many colum ns (n u m bered 1- 24). This fo rm at allow s co n v en tio n al m icrotitre plate acc esso ries such as m u ltich an n el pipettes and plate sh ak ers to be u sed w ith Q -plates, w hile red u cin g the sp ace req u irem en t o f any given clone lib rary by a facto r o f four. Since sto rag e o f clones at -70°C is a lim iting facto r the reduced space re q u irem en t is sig n ifican t. Q -plates have a total volum e o f
o f the volum e du rin g freezing. F ig u re 3-3 sh o w s a pho to g rap h o f a Q- plate.
3.2.4
Quality assessment by hybridisation
H igh d en sity clone arrays on n ylon m em branes w ere generated as d e s c rib e d in Materials and M et h o d s, u sin g a ro b o tic sp o ttin g machine develo p ed in the lab. C lones w ere arrayed at a den sity o f 2 0 ,7 3 6 per 22 cm X 22 cm m em brane (54 Q -p lates).
In o rd e r to a s se s s the q uality o f the cDNA lib rary , several h y b rid isatio n exp erim en ts w ere carried out w ith p ro b es o f g enes, w h o s e e x p re ss io n levels are k n o w n . The n u m b e r o f p o sitiv e ly h y b rid is in g clones, im m ediately indicates w h eth er a k n o w n sequ en ce is rep resen ted in the lib rary at the expected level. F ig u re s 3-4 - 3-8 show h y b rid isatio n resu lts w ith the fo llo w in g p ro b es: total hum an DNA, m ouse p -actin, rib o so m al gene, p o ly -A olig o and g ly c e ra ld e h y d e -3 -p h o s p h a te d e h y d ro g e n a se (G A P D H ).
The total human DNA p ro b e is expected to h y b rid ise w ith th o se cDNA clones co n tain ing com m on rep etitiv e seq u en ce elem ents (such as Alu sine elem ent), since the co m plexity o f the p ro b e is so high that single copy sequ en ces have in su ffic ie n t sp ecific activity to detect co m plem entary cDNA clo nes. The h y b rid isa tio n s h o w s that app ro xim a tely 1.5% o f clones h y b rid is e w ith total hum an DNA. This re su lt is c o n s is te n t w ith som e fin d in g s that s u g g e st betw een 1 - 2 %
e x p re ss e d p ro te in s contain Alu rep etitiv e elem ents (M ak alo w sk i et a l., 1994). A lthough Alu elem ents fo rm the m ajority o f rep etitiv e elem ents in the hum an genom e there are likely to be oth ers that give detectable sig nal w ith a total hum an DNA p ro be. W hile this h y b rid isatio n by no m eans gives a m easu re o f the nu m b e r o f tra n scrib ed rep eats, it does in d icate that the cDNA library does n o t contain an unex p ected ly large n u m b e r o f rep etitiv e elem en ts, a p ro b le m com m only en co u n tered w hen a s ig n ific a n t g enom ic DNA contam ination has occured.
^ %# »» % % # # % W v # % %% % % % % % % w %»^ #& » , % % . % % #.%%% V ^ #$*%% #.%$ %&. %, %L V ■%. V %. % - ^ V Vj%- ^ ^ 1* ^ ^ ^ %. %k > #, V * \ ' ^ m », ^ m . %/* sm « « , * ^ » w , * # " * '* '& '
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Fig. 3-3 A photograph of a 384-well microtitre plate (Q-plate), showing twice as many rows (A - P) and twice as many columns (1 - 24) as a conventional 96-well microtitre plate. Plates are made in two materials, polystyrene and polypropylene.
.
a , -' ' t . ' ' ' . - ». . • • • • « • • • • • • • • • . • • . » ' • • V - # \r
f # r • •Fig. 3-4 An autoradiograph of a hybridisation with a total human DNA probe on a high density nylon filter carrying 21,120 human foetal brain oDNA
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clones. 150 ng human DNA were labelled with [ P - a ] dATP by random priming and hybridised according to Church and Gilbert (1984). Approximately 2% of clones show a positive hybridisation signal.
Fig. 3-5 An autoradiograph of a hybridisation with a mouse actin DNA probe on a high density nylon filter carrying 21,1 20 human foetal brain cDNA clones. 50 ng DNA were labelled with [^^P-a] dATP by random priming and
• • •
%
# # * « • * • • # # * • • # * - • « • • • . # • • • • • • # • * i • « • * • X <-.
. . • • • • • • V*. • • . ; • * # » * # # # « F • • • ' ' • * • • «Fig. 3-6 An autoradiograph of a hybridisation with human ribosomal precursor g en e DNA probe on a high density nylon filter carrying 21,120
32
human foetal brain cDNA clones. 50 ng DNA were labelled with [ P - a ] dATP by random priming and hybridised according to Church and Gilbert (1984).
tffeF S S "' lû ï
* v # # .# # . 4 # ## ^ ## # * # *
Fig. 3-7 An autoradiograph of a hybridisation with oligo-dA DNA probe on a high density nylon filter carrying 21,120 human foetal brain cDNA clones.The average length of oligomers was 40 bp. 200 ng DNA were labelled with [^^P-yl ATP using T4 polynucleotide kinase and hybridised according to Church and Gilbert (1984).
• e
e • •
Fig. 3-8 An autoradiograph of a hybridisation with human glyceraldehyde 3-phosphate deh ydrogenase (GAPDH) DNA probe on a high density nylon filter carrying 21,120 human foetal brain oDNA clones. 50 ng
32
DNA were labelled with [ P - a ] dATP by random priming and hybridised according to Church and Gilbert (1984).
A m ouse P-actin cDNA p ro b e id entified many h o m o lo g o u s human cDNA clones in the library an expected resu lt in view o f the high hom ology betw een the m o u se and hum an P-actin genes. The ho m o lo g y w ithin the w h o le o f the actin gene family m akes it likely that the cDNA clones id en tified by the h y b rid isa tio n co m p rise a m ix tu re o f a - a c tin , p-actin and y-actin all o f w h ich are actively tra n sc rib e d in brain tissu e. No attem pts w ere m ade to ch aracterise the actin clones fu rth e r and assig n them to su b -fam ily g ro u p s. H y b rid is a tio n s w ith both th e ribosom al RN A (rR N A ) p r e c u rs o r gene and the p o ly -A o ligo indicate th at w hile there are m any h y b rid is in g clones w ith b oth p ro b es the n u m b ers are not so high as to th ro w into do u b t the com plexity o f the library. T w o o f the m ost com m on sh o rtco m in g s o f cDNA libraries are that they either contain a very high p ro p o rtio n o f rR N A derived clones due to rR N A p rim ing d u rin g first strand cDNA sy n th e s is (a p ro b le m generally asso ciated more w ith cDNA libraries generated by ran d o m hexam er p rim in g o f mRNA) or a high p ro p o rtio n o f clones th at contain alm o st e x clu siv ely po ly-d A t r a c t s .
The G APDH p ro b e identified ap p ro x im a tely 0 .3 % o f cDNA clones in the lib rary , a re su lt in line w ith the fact that the g ly c e ra ld e h y d e -3 -p h o s p h a te d eh y d ro g e n a se enzym e carries o u t one o f the steps in one o f the m o st active catabolic p ath w ay s in brain tiss u e , g ly c o ly sis . A s u b se t o f the G APDH clones w as an alysed fu rth e r by PCR am plification o f the cDNA in se rts. F ig u re 3-9 s h o w s the P C R p ro d u cts ru n on an ag aro se gel. The length o f the G A PDH com plete cod ing seq u en ce, extracted from the G e n b an k d atab ase, is 1 ,2 68 bp. The gel s h o w s that 71% o f the clones are ap p ro x im a te ly 1 ,2 00 bp in length and th at none are longer. This s u g g e s ts that 71% o f the G A PDH clones are full length and that none o f th e clo n es an alysed are derived fro m u n sp liced n u clear R N A m olecules. W hile it is tem pting to specu late that ap p rox im ate ly 70% o f 1 ,2 0 0 bp gen es are rep resen ted as full length in the cDNA lib rary , this as su m p tio n can n ot be m ade given v ariable clo n in g efficien cies o f
T: ' - ‘ ^ h' ' . . ^ ' ' . I . ^ - , 11 L : • ' - , ^ --i ^ t : a L CÜNA : "' r: r c p r c r c h W f l o : ) 6A d C 0 B 1 f l * s i ! y ; I . 1- i r-nm kralm ;■ n > ' . • ■■ V • ,c bp 1 2 6 4 1 2 3 4 5 6 7 8 91011 U 13 14 15 15 17 18 19 20 21 22 23 24 25 26 27 28 29 "T- r., 12 6 4
Fig. 3-9 A 1% agarose gel of PCR amplifications carried out on 34 clones hybridising positively with GAPDH probe. 5 pi of each PCR reaction were loaded in each lane. The size markers used were BsfEII digested lambda DNA,
The overall a s se s sm e n t o f the hum an foetal brain cDNA library based upon the tests d isc u s s e d above, is that its rep resen tatio n and com plexity are in line w ith expectation for a lib rary c o n stru cted fro m human brain tissue.
The lib rary has been w idely d istrib u te d in fo rm o f high d ensity filter array s to 56 labs and 2 8 3 0 p otential p o s itiv e clones have been identified.