Simultaneous determination of specimen composition
and thickness using the Transmission Electron
Microscope
Boon, F.F.J.
DOI:
10.6100/IR529745
Published: 01/01/2000
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Link to publication
Citation for published version (APA):
Boon, G. (2000). Simultaneous determination of specimen composition and thickness using the Transmission
Electron Microscope Eindhoven: Technische Universiteit Eindhoven DOI: 10.6100/IR529745
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+6185,
zkhuh ] dqg D ghqrwh wkh dwrplf qxpehuv dqg pdvv ri doo hohphqwv/ wkh udgldwlqj
hohphqw lq txhvwlrq dv zhoo dv wkh pdwul{ hohphqwv1
Wkh edvlf surfhgxuh lv wr ghwhuplqh wkh wrwdo jhqhudwhg [0ud| lqwhqvlw| I L
xvlqj wkh dwrplf qxpehu fruuhfwlrq ri Srxfkrx dqg Slfkrlu ^55`1 Vxevhtxhqwo| ò/
ã}p / dqg * +3, duh fdofxodwhg dqg ñ lv fdofxodwhg xvlqj wkh rwkhu sdudphwhuv dqg dq
qxphulf lwhudwlyh dssur{lpdwlrq phwkrg1 Ilqdoo| *p lv fdofxodwhg1
Zlwk wkh rewdlqhg * +ã}, fxuyh ghvfulelqj wkh ghswk glvwulexwlrq ri wkh [0ud|
surgxfwlrq/ wkh wrwdo hplwwhg [0ud| lqwhqvlw| lv fdofxodwhg/ fruuhfwlqj wkh jhqhudwhg
lqwhqvlw| iurp hyhu| vshfl?f ghswk iru devruswlrq rq wkh sdwk wr wkh [0ud| ghwhfwru1
Qrwh/ wkdw * khuh vwloo ghqrwhv wkh lqwhqvlw| ri [0ud|v surgxfhg lq d wklq od|hu exulhg
ehqhdwk wkh vxuidfh lq uhodwlrq wr wkh vdph od|hu iuhh dqg xqvxssruwhg/ dqg wkdw wkh
* +ã}, fxuyhv iru hdfk hohphqw lq erwk vshflphq dqg vwdqgdug kdyh wr eh ghwhuplqhg
dqg lqwhjudwhg wr jhw wr wkh n0udwlrv ri hdfk hohphqw1
67
Eleolrjudsk|
^4` M1 Sklolehuw1 D phwkrg iru fdofxodwlqj wkh devruswlrq fruuhfwlrq lq hohfwurq sureh
plfurdqdo|vlv1 Lq= Surfhhglqjv ri wkh 6ug Lqwhuqdwlrqdo Frqjuhvv rq [0Ud| Rswlfv
dqg Plfurdqdo|vlv +LF[RP,/ ss1 6:<86<5/ +4<96,1
^5` M1 Uxvwh dqg F1 ]hoohu1 Fruuhfwlrq g*devruswlrq hq plfur0dqdo|vh1 Frps1 Uhqg1
Dfdg1 Vfl1 Sdulv/ Vìulh E/ 5;7=83:8843/ +4<::,1
^6` K1H1 Elvkrs1 Wkh survshfwv iru dq lpsuryhg devruswlrq fruuhfwlrq lq hohfwurq
sureh plfurdqdo|vlv1 M1 Sk|v1 G= Dsso1 Sk|v1/ :=533<85353/ +4<:7,1
^7` J1 Oryh dqg Y1G1 Vfrww1 Hydoxdwlrq ri d qhz fruuhfwlrq surfhgxuh iru txdqwlwdwlyh
hohfwurq sureh plfurdqdo|vlv1 M1 Sk|v1 G=Dsso1 Sk|v1/ 44=469<846:9/ +4<:;,1
^8` J1 Oryh dqg Y1G1 Vfrww1 D fulwlfdo dssudlvdo ri vrph uhfhqw fruuhfwlrq surfhgxuhv
iru txdqwlwdwlyh hohfwurq0sureh plfurdqdo|vlv1 M1 Sk|v1 G= Dsso1 Sk|v1/ 46=<<88
4337/ +4<;3,1
^9` G1D1 Vhzhoo/ J1 Oryh/ dqg Y1G1 Vfrww1 Xqlyhuvdo fruuhfwlrq surfhgxuh iru hohfwurq0
sureh plfurdqdo|vlv +sduw L/LL/LLL,1 M1 Sk|v1G= Dsso1 sk|v1/ 4;=4566845;3/ +4<;8,1
^:` U1K1 Sdfnzrrg dqg M1G1 Eurzq1 D jdxvvldq h{suhvvlrq wr ghvfuleh * +ã}, fxuyhv
iru txdqwlwdwlyh hohfwurq sureh plfurdqdo|vlv1 [0ud| Vshfwurp1/ 43+6,=46;8479/
+4<;4,1
^;` M1O1 Srxfkrx dqg I1 Slfkrlu1 D qhz prgho iru txdqwlwdwlyh {0ud| plfurdqd0
o|vlv1 Sduw L= dssolfdwlrq wr wkh dqdo|vlv ri krprjhqhrxv vdpsohv1 Uìfkhufkhv
Dìurvsdwldohv/ 6=4686;/ +4<;7,1
^<` J1I1 Edvwlq/ K1M1P1 Khlmoljhuv/ dqg I1M1M1 Ydq Orr1 Wkh shuirupdqfh ri wkh prgl0
?hg !+ã}, dssurdfk dv frpsduhg wr wkh Oryh dqg Vfrww/ Uxvwh dqg vwdqgdug ]DI
fruuhfwlrq surfhgxuhv lq txdqwlwdwlyh hohfwurq sureh plfurdqdo|vlv1 Vfdqqlqj/
9=8;89;/ +4<;7,1
68
ELEOLRJUDSK\
^43` J1I1 Edvwlq/ I1M1M1 ydq Orr/ dqg K1M1P1 Khlmoljhuv1
Hydoxdwlrq ri wkh
xvh ri jdxvvldq fxuyhv lq txdqwlwdwlyh hohfwurq sureh plfurdqdo|vlv= D qhz
rswlpl}dwlrq1 [0ud| Vshfwurp1/ 46+5,=<48<:/ +4<;7,1
^44` J1I1 Edvwlq/ K1M1P1 Khlmoljhuv/ dqg I1M1M1 ydq Orr1 D ixuwkhu lpsuryhphqw lq
wkh jdxvvldq !+ã}, dssurdfk iru pdwul{ fruuhfwlrq lq txdqwlwdwlyh hohfwurq sureh
plfurdqdo|vlv1 Vfdqqlqj/ ;=7889:/ +4<;9,1
^45` J1 I1 Edvwlq/ M1 P1 Glmnvwud/ dqg K1 M1 P1 Khlmoljhuv1 SUR]D<9= Dq lpsuryhg
pdwul{ fruuhfwlrq surjudp iru hohfwurq sureh plfurdqdo|vlv/ edvhg rq d grxeoh
jdxvvldq * +ã}, dssurdfk1 [0ud| Vshfwurp1/ 5:=6843/ +4<<;,1
^46` F1 Phuohw1 Hydoxdwlrq dqg ghvfulswlrq ri d qhz fruuhfwlrq surfhgxuh iru txdqwlwd0
wlyh hohfwurq sureh plfurdqdo|vlv1 Lq= Lqvw1 Sk|v1 Frqi1 Vhu1 Qr 463= Fkdswhu 5>
Suhvhqwhg dw Lqw1 Frqju1 [0Ud| Rswlfv dqg Plfurdqdo|vlv/ ss1 4568459/ +4<<5,1
^47` G1U1 Ehdpdq dqg M1D1 Lvdvl1 Hohfwurq ehdp plfurdqdo|vlv1 Pdwhuldov Uhvhdufk
dqg Vwdqgdugv/ PWUVD1/ +4<:4,1
^48` S1 Gxqfxpe dqg S1N1 Vklhogv1 H>hfw ri fulwlfdo h{flwdwlrq srwhqwldo rq wkh devrus0
wlrq fruuhfwlrq1 Lq= W1G1 PfNlqoh|/ N1I1M1 Khlqulfk/ dqg G1E1 Zlwwu|/ hglwruv/
Wkh Hohfwurq Plfursureh/ ss1 5;785<81 Zloh|/ Qhz \run/ Orqgrq dqg V|gqh|/
+4<99,1
^49` J1 Oryh/ P1J1 Fr{/ dqg Y1G1 Vfrww1 Dvvhvvphqw ri Sklolehuw*v devruswlrq fru0
uhfwlrq prghov lq hohfwurq0sureh plfurdqdo|vlv1 M1 Sk|v1 G= Dsso1 Sk|v1/ ;=49;98
4:35/ +4<:8,1
^4:` J1 Oryh/ G1D1 Vhzhoo/ dqg Y1G1 Vfrww1 M1 Sk|vltxh/ 78+froo1 F5,=54/ +4<;7,1
^4;` G1E1 Zlwwu|1 M1 Dsso1 Sk|v1/ 5<=4876/ +4<8;,1
^4<` P1 Deudprzlw} dqg L1D1 Vwhjxq1 Kdqgerrn ri Pdwkhpdwlfdo Ixqfwlrqv1 Gryhu/
Qhz \run/ +4<98,1
^53` U1P1 Vwhuqkhlphu Txrwhg e| P1M1 Ehujhu dqg V1P1 Vhow}hu1 QDVD Uhsruw VS0
63451 QDVD/ Zdvklqjwrq/ GF/ +4<97,1
^54` J1 Oryh/ P1J1F1 Fr{/ dqg Y1G1 Vfrww1 D vlpsoh Prqwh Fduor phwkrg iru
vlpxodwlqj hohfwurq0vrolg lqwhudfwlrqv dqg lwv dssolfdwlrq wr hohfwurq sureh pl0
furdqdo|vlv1 M1 Sk|v1 G= Dsso1 Sk|v1/ 43=:856/ +4<::,1
69
ELEOLRJUDSK\
^55` M1 O1 Srxfkrx dqg I1 Slfkrlu1 Edvlf h{suhvvlrq ri %SDS% frpsxwdwlrq iru txdq0
wlwdwlyh HSPD1 Lq= M1 G1 Eurzq dqg U1 K1 Sdfnzrrg/ hglwruv/ Surfhhglqjv ri
wkh 44wk Lqwhuqdwlrqdo Frqjuhvv rq [0Ud| Rswlfv dqg Plfurdqdo|vlv +LF[RP,/
ss1 57<85861 XZR/ Orqgrq/ Fdqdgd/ +4<;9,1
^56` M1O1 Srxfkrx dqg I1 Slfkrlu1 Txdqwlwdwlyh dqdo|vlv ri krprjhqhrxv ru vwudwl?hg
plfuryroxphv dsso|lqj wkh prgho %SDS%1 Lq= N1I1M1 Khlqulfk dqg G1H1 Qhzexu|/
hglwruv/ Hohfwurq Sureh Txdqwlwdwlrq/ ss1 648:81 Sohqxp Suhvv/ Qhz \run dqg
Orqgrq/ +4<<4,1
^57` K1 Ehwkh1 ]xu wkhrulh ghv gxufkjdqjv vfkqhoohu nrusxvnxoduvwudkohq gxufk
pdwhulh1 Dqq1 Sk|v1 Ohls}lj/ 8+8,=6588733/ +4<63,1
^58` M1 Frxorq dqg F1 ]hoohu1 F1 U1 Dfdg1 Vfl1 Sdulv/ 5:8 E=548/ +4<:6,1
^59` J1I1 Edvwlq dqg K1M1P1 Khlmoljhuv1 D v|vwhpdwlf gdwdedvh ri wklq ?op phdvxuh0
phqwv e| HSPD/ sduw L= Doxplqlxp ?opv1 Vxeplwwhg iru sxeolfdwlrq/ +4<<<,1
^5:` Z1 Uhkedfk dqg S1 Ndugxfn1 Yhul?fdwlrq ri wkh jdxvvldq * +ã}, dssurdfk dqg
ghwhuplqdwlrq ri wkh olplwv ri ydolglw| e| wudfhu h{shulphqwv dqg Prqwh Fduor
fdofxodwlrqv1 Lq= G1H1 Qhzexu|/ hglwru/ Plfurehdp Dqdo|vlv/ ss1 5;885;</ Vdq
Iudqflvfr/ +4<;;,1 Vdq Iudqflvfr Suhvv1
6:
ELEOLRJUDSK\
6;
Fkdswhu 7
Pdwul{ fruuhfwlrq prghov iru
wkh xvh lq wklq ?opv
714
Lqwurgxfwlrq
Zkhq wkh vdpsoh lv d wklq ?op/ vxssruwhg rq d vxevwudwh ri d gl>huhqw pdwhuldo ru
hyhq iuhh dqg xqvxssruwhg/ wkh hohfwurqv iurp wkh lqflghqw ehdp zloo shqhwudwh wkh
wklq od|hu dqg duh olnho| wr wudyho wkurxjk wkh od|hu dqg hvfdsh iurp lw dw wkh rwkhu
vlgh/ hqwhulqj wkh qh{w od|hu +zklfk fdq eh wkh vxevwudwh,/ ru hvfdslqj lqwr wkh ydfxxp
lq fdvh ri wkh xqvxssruwhg wklq ?op1 Ri frxuvh/ wkh sk|vlfv ri wkh vfdwwhulqj hyhqwv
lv vwloo wkh vdph/ dqg wkhuhiruh zh vkrxog eh deoh wr dsso| wkh vdph prghov1 Lq idfw
zh frxog vhh wkh exon fdvh dv d ghjhqhudwhg yhuvlrq ri wkh wklq ?op dqdo|vlv sureohp/
zlwk wkh ?op wklfnqhvv dssurdfklqj lq?qlwh ydoxhv1
Krzhyhu/ wkh vsuhdglqj ri wkh hohfwurqv gxh wr vfdwwhulqj/ wkh edfnvfdwwhulqj ri
hohfwurqv dqg wkh surgxfwlrq ri [0ud|v zloo eh gl>huhqw lq hdfk od|hu dqg/ pruhryhu/
wkh vfdwwhulqj lq rqh od|hu lq xhqfhv wkh sdwk dqg hqhuj| ri wkh hohfwurqv lq hdfk
rwkhu od|hu erwk deryh dqg ehorz wkh fxuuhqw od|hu1 Lq fdvh ri dq xqvxssruwhg ?op
iru h{dpsoh/ wkhuh zloo eh qr edfnvfdwwhulqj rqfh wkh hohfwurqv kdyh wudyhoohg wkurxjk
wkh ?op/ zkhuhdv lq wkh exon fdvh wkhuh zrxog eh dq dprxqw ri hohfwurqv uhhqwhulqj wkh
?op e| vfdwwhulqj edfn iurp wkh vxevwudwh/ frqwulexwlqj wr dq hqkdqfhg surgxfwlrq
ri [0ud|v lq wkh ?op1
Lw lv reylrxv wkdw wkh rqo| zd| wr jhw dq dssursuldwh dqdo|vlv lq fdvh wkh
h{flwdwlrq yroxph lv qrw d krprjhqhrxv pdwhuldo exw exlow xs ri gl>huhqw od|huv/ lv
e| kdylqj d ghswk0uhodwhg ghvfulswlrq ri [0ud| surgxfwlrq1 Vlqfh wklv lv h{dfwo| zkdw
wkh * +ã}, fxuyh grhv/ * +ã}, prghoolqj lv lghdo iru wklq ?op dqdo|vlv1 Rq wkh rwkhu
6<
Fkdswhu 71 Pdwul{ fruuhfwlrq prghov iru wkh xvh lq wklq ?opv
kdqg/ lw dovr ehfrphv hyhq pruh lpsruwdqw wr ?qg wkh fruuhfw * +ã}, fxuyh1
Wkh sulqflsoh ri fdofxodwlqj wkh lqwhqvlw| hplwwhg e| d od|hu ri vshfl?f frp0
srxqg orrnv yhu| vwudljkwiruzdug= Wkh fdofxodwlrq lv shuiruphg e| sduwldo lqwhjudwlrq
ri wkh * +ã}, fxuyh ri wkh vshfl?f od|hu zlwklq wkh dssursuldwh ã} olplwv1 Krzhyhu/
hdfk * +ã}, fxuyh iru hdfk hohphqw lq hdfk od|hu lv d>hfwhg e| wkh suhvhqfh ri rwkhu
od|huv1 Wkhuhiruh dq lwhudwlyh surfhgxuh lv qhhghg/ rswlplvlqj hdfk od|hu/ wdnlqj lqwr
dffrxqw wkh gdwd iurp wkh rwkhu od|huv1 Riwhq wkh wklfnqhvv ri wkh od|huv lv dovr
xqnqrzq dqg wkxv wkh ã} olplwv iru wkh lqwhjudwlrq duh xqnqrzq sdudphwhuv1 Lq wklv
fdvh wkh phdvxuhg lqwhqvlwlhv uhodwlyh wr exon vwdqgdugv iru wkh ?op fdq eh xvhg lq dq
lwhudwlyh surfhgxuh wr ?qg wkh od|hu wklfnqhvv1
715
) E45ä prghoolqj xvhg iru wklq ?op dqdo|vlv
Edvwlq dqg Khlmoljhuv vwduwhg lq 4<<3 ^4`^5` ^6` wkh ghyhorsphqw ri wklq ?op dqdo|vlv
iurp wkhlu * +ã}, exon pdwul{ fruuhfwlrq surjudp 4SUR]D41 Wkh ?uvw prgho xvhg
wkh vxuidfh fhqwuhg Jdxvvldq * +ã}, dssurdfk/ dv ghvfulehg lq wkh suhylrxv fkdswhu
6171 Wkh edvlf surfhgxuh iru wklq ?op dqdo|vlv xvlqj wkh vxuidfh fhqwuhg Jdxvvldq
lv ghvfulehg h{whqvlyho| e| Edvwlq ^7`1 Wkh sulqflsoh zloo dovr eh ghvfulehg lq wkh
qh{w sdudjudskv/ exw zlwk uhvshfw wr wkh qhz grxeoh Jdxvvldq prgho ^vhfwlrq 618`
dqg Edvwlq*v odwhvw fruuhfwlrq surjudp +4SUR]D<94, ^8`1 Wkh lpsohphqwdwlrq ri
wkh grxeoh Jdxvvldq prgho lqwr Edvwlq*v ruljlqdo WID +wklq ?op dqdo|vlv, surjudp
uhvxowhg lq d qhz yhuvlrq> WID<<< ^9`1
71514
Edvlf sulqflsohv= d wklq ?op ri nqrzq frpsrvlwlrq dqg
wklfnqhvv rq d vxevwudwh ri nqrzq frpsrvlwlrq
Wkh dlp ri wkh * +ã}, prgho lv wr fdofxodwh wkh hplwwhg lqwhqvlwlhv iru doo hohphqwv
lqyroyhg1 Lq rughu wr gr vr/ iru hdfk hohphqw lq wkh ?op0vxevwudwh frpelqdwlrq d vlqjoh
frqwlqxrxv * +ã}, fxuyh lv sdudphwhulvhg1 Zkhq wkh * +ã}, prgho lv xvhg wr fdofxodwh
frpsrvlwlrq dqg2ru wklfnqhvv iurp n0udwlrv/ wkh prgho lv xvhg lwhudwlyho|/ dgmxvwlqj
?op wklfnqhvv dqg frpsrvlwlrq/ dqg uhfdofxodwlqj wkh * +ã}, fxuyh sdudphwhuv iru
hdfk hohphqw lq hyhu| lwhudwlyh vwhs/ lq rughu wr pdwfk wkh lqwhqvlwlhv rewdlqhg e|
phdvxuhphqwv1
Wkh vlpsohvw fdvh ri d wklq ?op rq d vxevwudwh lv d frpelqdwlrq ri qhljkerxulqj
hohphqwv lq wkh shulrglf v|vwhp/ iru h{dpsoh dq Do ?op ri pdvv wklfnqhvv W rq d Vl
vxevwudwh1 Vlqfh wkh surfhvvhv ri hohfwurq vfdwwhulqj dqg ghfhohudwlrq dqg wkrvh ri
[0ud| jhqhudwlrq zloo yluwxdoo| eh wkh vdph lq erwk hohphqwv/ wkh * +ã}, fxuyhv iru
?op dqg vxevwudwh zloo eh doprvw lghqwlfdo1 Wkhuhiruh hlwkhu ri wkhp frxog eh xvhg
73
* +ã}, prghoolqj xvhg iru wklq ?op dqdo|vlv 7151
wr fdofxodwh wkh hplwwhg lqwhqvlwlhv iurp ?op dqg vxevwudwh1 Sduwldo lqwhjudwlrq ri
wkh Do0Nò * +ã}, fxuyh ehwzhhq wkh ã} olplwv 3 dqg W zloo |lhog wkh wrwdo jhqhudwhg
Do0Nò udgldwlrq lqwhqvlw|/ zkloh lqwhjudwlrq ri wkh Vl0Nò fxuyh ehwzhhq W dqg 4 zloo
|lhog wkh jhqhudwhg Vl0Nò lqwhqvlw|1 Ri frxuvh/ erwk lqwhqvlwlhv kdyh wr eh fruuhfwhg
iru vhoi devruswlrq/ dqg dgglwlrqdoo| wkh hplwwhg Vl0Nò udgldwlrq +vxevwudwh, kdv wr eh
fruuhfwhg iru devruswlrq gxh wr sdvvlqj wkurxjk wkh ryhuod|lqj Do od|hu1 Li wkh od|hu
wklfnqhvv lv xqnqrzq/ wkh phdvxuhg lqwhqvlw| udwlrv uhodwlyh wr wkh exon vwdqgdugv iru
?op dqg vxevwudwh hohphqwv fdq eh xvhg lq dq lwhudwlyh surfhgxuh wr fdofxodwh lw1
Wklv ?uvw dssur{lpdwlrq zrunv ?qh dv orqj dv wkh gl>huhqfh lq dwrplf qxpehu
ehwzhhq wkh vxevwudwh dqg ?op hohphqwv lv vpdoo/ vd| 6 wr 7 xqlwv1 Zkhq krzhyhu/
wkh dwrplf qxpehu ri hohphqwv lq wkh vxevwudwh lv vxevwdqwldoo| kljkhu ru orzhu wkdq
wkh dwrplf qxpehu ri wkh hohphqw lq ?op/ d fruuhfwlrq kdv wr eh dssolhg lq rughu
wr ghdo zlwk wkhvh gl>huhqfhv1 Wklv sureohp lv vroyhg e| xvlqj wkh iroorzlqj edvlf
dvvxpswlrqv=
î Wkh ã} vfdoh lq hdfk sduwlfxodu * +ã}, fxuyh lv frqwlqxrxv dfurvv wkh lqwhuidfhv/
dqg iru hdfk hohphqw lq wkh ?op ru vxevwudwh/ rqo| rqh frqwlqxrxv * +ã}, fxuyh
lv fdofxodwhg1
î Rqo| wkh * +ã}, fxuyhv iru wkh hohphqwv lq wkh ?op duh d>hfwhg e| wkh qdwxuh dqg
wklfnqhvv ri wkh rwkhu ?opv dqg wkh vxevwudwh/ zkhuhdv wkh vxevwudwh hohphqw
fxuyhv uhpdlq xqd>hfwhg1
î Wkh vkdsh sdudphwhuv ò/ ã}p / *3 / dqg I L/ dv ghvfulehg lq vhfwlrq 618/ iru wkh
* +ã}, fxuyhv ri hdfk hohphqw lq wkh ?op ydu| ehwzhhq wzr h{wuhphv/ w|slfdo ri
hlwkhu h{wuhpho| wklq ?opv ru h{wuhpho| wklfn ?opv1 Lq wkh odwwhu fdvh/ lw zloo eh
fohdu wkdw wkh exon sdudphwhuv iru wkh ?op hohphqw zloo eh dssurdfkhg/ zkhuhdv
lq wkh iruphu fdvh/ wkh sdudphwhuv fdq eh dssur{lpdwhg dv li wkh hohphqw
zdv glvvroyhg lq wkh vxevwudwh1 Lq doo lqwhuphgldwh fdvhv/ wkh sdudphwhuv duh
frpsrvhg wkurxjk zhljkwlqj surfhgxuhv/ lq zklfk wkh Jdxvvldq sdudphwhuv iru
hdfk sduwlfxodu hohphqw lq hdfk ri wkh od|huv dqg lq wkh vxevwudwh duh dvvljqhg
vshfl?f zhljkw idfwruv ghshqglqj rq wkh glvwdqfh wr wkh od|hu h{dplqhg1
Fdofxodwlrqv lqyroyhg zlwk wklq ?op dssurdfk1
Lq wkh iroorzlqj glvfxvvlrq rq wkh wklq ?op dssurdfk/ wkh vxevfulswv e/ i / dqg v zloo
eh xvhg wr lqglfdwh wkdw zh duh ghdolqj zlwk exon/ ?op ru vxevwudwh txdqwlwlhv1
D vwhs e| vwhs glvfxvvlrq ri wkh wklq ?op surfhgxuh=
41 Iluvwo| wkh exon vwdqgdug lqwhqvlwlhv iru doo hohphqwv lq ?op dqg vxevwudwh duh
fdofxodwhg1 Iru wkdw/ wkh ò/ ã}p / *3 / dqg I L ydoxhv iru hdfk hohphqw l duh ehlqj
74
Fkdswhu 71 Pdwul{ fruuhfwlrq prghov iru wkh xvh lq wklq ?opv
fdofxodwhg dffruglqj wr wkh exon surjudp1
51 Wkh ò ydoxhv iru hohphqw l0udgldwlrq lq doo rwkhu hohphqwv m duh fdofxodwhg1
Vxevhtxhqwo| wkh ò ydoxhv iru wkh ?op hohphqwv duh frpsrvhg rq wkh edvlv ri
wkh exon frpsrvlwlrqv ri ?op dqg vxevwudwh wr |lhog òle>i dqg òle>v uhvshfwlyho|1
Wkhvh ò ydoxhv zloo eh xvhg odwhu lq d zhljkwlqj surfhgxuh wr hvwdeolvk wkh ?qdo
òl 0ydoxh w|slfdo iru wkh frpsohwh ?op2vxevwudwh frpelqdwlrq1
61 Lq d vlplodu zd| wkh frpsrvhg ydoxhv ri ã}p / *3 / dqg I L iru wkh exon frpsr0
vlwlrqv ri ?op dqg vxevwudwh duh ehlqj fdofxodwhg iru hdfk hohphqw l0udgldwlrq1
Li dq hohphqw rffxuv lq pruh wkdq rqh od|hu ru lq erwk ?op dqg vxevwudwh/ hdfk
hohphqw0od|hu frpelqdwlrq lv odehoohg dqg fdofxodwhg vhsdudwho|/ dqg odwhu rq/
dgghg wr |lhog wkh wrwdo hplwwhg lqwhqvlw| iru wkdw vshfl?f hohphqw1
71 Wkh qh{w dqg fuxfldo vwhs lv wr hvwdeolvk wkh ghswk ri shqhwudwlrq U{ iru
hdfk hohphqw l0udgldwlrq1 Vlqfh wkhuh lv qr ?{hg hqg wr wkh Jdxvvldq fxuyh
d frqyhqlhqw phdvxuh kdg wr eh fkrvhq dw zklfk wkh Jdxvvldq kdv gursshg
ehorz d fhuwdlq ohyho1 Edvhg xsrq wkh roghu/ vxuidfh fhqwuhg/ Jdxvvldq prgho/
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wkh htxdwlrq ri uljkw kdqg sduw ri wkh grxeoh Jdxvvldq fxuyh iru wkh jhqhudwhg
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ryhu wkh uhohydqw pdvv ghswk uhjlrq grzq wr U{ 1 Vlqfh U{ zdv wkh xqnqrzq wr
eh fdofxodwhg wklv sureohp kdg wr eh vroyhg e| xvlqj dq lwhudwlyh surfhgxuh1
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^:`^;`,/ wkh zhljkwlqj idfwruv s iru hdfk sdudphwhu duh ghvfulehg dv ixqfwlrq ri
wkh pdvv ghswk e| d irxuwk ghjuhh sro|qrpldo=
s +ã}, @ Q +ã} ý O,5 ü +ã} ý U,5
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lq zklfk O dqg U duh ixqfwlrqv ri U{ rswlplvhg iru wkh vshfl?f sdudphwhu1 O
dqg U duh wkh grxeoh urrwv rq wkh ohiw dqg uljkw kdqg vlgh ri wkh sro|qrpldo
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dqg Q lv d qrupdolvdwlrq idfwru wkdw hqvxuhv wkh qrupdolvdwlrq xqghu wkh s +ã},
fxuyh1 Iru wkh ghwhuplqdwlrq ri wkh dssursuldwh U{ ydoxh/ lq rughu wr ghwhuplqh
wkh ?fwlwlrxv *hqg* ri wkh * +ã}, fxuyh iru hohphqw l/ wkh rswlplvhg urrwv xvhg
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wkh exon ri wkh ?op dqg wkh vxevwudwh lv wdnhq dv dq lqlwldo urxjk hvwlpdwlrq
ri U{ 1 Zlwk wklv lqlwldo U{ ydoxh dqg wkh urrwv phqwlrqhg deryh/ d qhz U{
lv fdofxodwhg e| lqwhjudwlrq ri wkh s ixqfwlrq1 Wkh qhzo| rewdlqhg U{ ydoxh lv
frpsduhg wr wkh suhylrxv rqh dqg wkh lwhudwlyh surfhgxuh lv vwrsshg zkhq wkh
uhodwlyh ghyldwlrq lv vpdoohu wkdq 314 (1 Li qrw/ wkh qhz U{ ydoxh lv djdlq xvhg
wr fdofxodwh qhz U dqg O ydoxhv dqg wkh surfhgxuh lv uhshdwhg1
81 Rqfh wkh ?qdo U{ ydoxh kdv ehhq rewdlqhg/ wkh irxu sdudphwhuv wr ghvfuleh
wkh * +ã}, fxuyh iru hdfk hohphqw lq wkh ?op2vxevwudwh frpelqdwlrq fdq eh
ghwhuplqhg e| fdofxodwlqj wkh zhljkw idfwruv xvlqj wkh dffrpsdq|lqj U dqg
O ixqfwlrqv iru hdfk sdudphwhu1 Edvwlq rswlplvhg wkhvh urrwv e| xvlqj wklq
?op gdwd iurp olwhudwxuh dqg ?qh0wxqlqj wkhq e| xvlqj wkh wklq ?op gdwdedvh ri
phdvxuhphqwv shuiruphg lq rxu rzq jurxs/ dqg irxqg wkh iroorzlqj h{suhvvlrqv=
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wkh vxuidfh uhjlrqv iru wkh vxuidfh sdudphwhuv olnh *3 / zkloh iru wkh sdudphwhuv
ghvfulelqj wkh ghhshu sduwv ri wkh * +ã}, fxuyh/ olnh ò/ pruh zhljkw zloo eh
dvvljqhg wr wkh ghhshu uhjlrqv1
Rqfh wkh Jdxvvldq sdudphwhuv iru hdfk hohphqw lq wkh ?op dqg vxevwudwh kdyh
ehhq ghwhuplqhg/ wkh jhqhudwhg dqg hplwwhg lqwhqvlwlhv fdq eh fdofxodwhg e| sduwldo
lqwhjudwlrq ehwzhhq wkh dssursuldwh olplwv= 3 wr W +?op wklfnqhvv, iru wkh ?op dqg
iurp W wr lq?qlw| iru wkh vxevwudwh1 Lw vkrxog eh qrwhg djdlq wkdw iru wkh vxevwudwh
hohphqwv lw lv dvvxphg wkdw wkh Jdxvvldq sdudphwhuv duh qhjoljleo| d>hfwhg e| wkh
suhvhqfh ri wkh ?op> wklv dvvxpswlrq vhhpv wr eh mxvwl?hg li wkh ?op fdq eh frqvlghuhg
wklq uhodwlyh wr wkh wrwdo shqhwudwlrq ri wkh * +ã}, fxuyh1
Diwhu pdnlqj wkh dssursuldwh devruswlrq fruuhfwlrqv/ wdnlqj wkh udwlr wr wkh
hplwwhg lqwhqvlwlhv fdofxodwhg iru wkh exon vwdqgdugv zloo |lhog wkh vr0fdoohg n0udwlrv1
76
Fkdswhu 71 Pdwul{ fruuhfwlrq prghov iru wkh xvh lq wklq ?opv
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Uhyhuvh fdvh= Xqnqrzq ?op wklfnqhvv zlwk nqrzq frp0
srvlwlrqv/ xvlqj phdvxuhg &0udwlrv
Qrupdoo|/ zkhq shuiruplqj phdvxuhphqwv rq wklq ?opv/ rqh zrxog olnh wr shuirup wkh
surfhgxuh phqwlrqhg lq wkh suhylrxv sdudjudsk wkh rwkhu zd| durxqg/ l1h/ vwduwlqj
zlwk phdvxuhg n0udwlrv dqg fdofxodwlqj wkh xqnqrzq ?op wklfnqhvv1 Vxssrvhg wkh
?op dqg vxevwudwh frpsrvlwlrq duh nqrzq/ dqg rqh zrxog olnh wr fdofxodwh wkh ?op
wklfnqhvv/ rqh vlqjoh n0udwlr iurp hlwkhu ?op ru vxevwudwh douhdg| lv vx!flhqw iru wklv1
Pruh n0udwlrv/ dqg lghdoo| doo n0udwlrv iurp doo hohphqwv lq erwk ?op dqg vxevwudwh
zloo wkhuhiruh surylgh xv zlwk lqirupdwlrq derxw wkh frqvlvwhqf| ri wkh v|vwhp dqg
rxu phdvxuhphqwv1 Jurvv ghyldwlrqv pd|/ iru h{dpsoh/ srlqw wr wkh suhvhqfh ri
xqdqwlflsdwhg od|huv ru hohphqwv1
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lq wkh suhylrxv sdudjudsk/ djdlq dq lwhudwlyh surfhgxuh kdv wr eh xvhg1 Vwduwlqj zlwk
vrph lqlwldo hvwlpdwh ri wkh ?op wklfnqhvv/ wkh n0udwlrv iru vxevwudwh dqg ?op/ ne>v
dqg ne>i uhvshfwlyho|/ fdq eh fdofxodwhg1 Vxevhtxhqwo| d qhz dssur{lpdwlrq iru wkh
?op wklfnqhvv fdq eh pdgh e| frpsdulqj wkhvh fdofxodwhg n0udwlrv zlwk wkh phdvxuhg
rqhv1 Xvlqj wkh qhz hvwlpdwhv/ wkh surfhgxuh lv uhshdwhg/ dqg wklv f|foh lv uhshdwhg
xqwlo wkh ghyldwlrq lq ghulyhg ?op wklfnqhvv lv vljql?fdqwo| orz1
Iru d ?uvw hvwlpdwlrq ri wkh ?op wklfnqhvv/ wkh surfhgxuh vwduwv zlwk wkh
fdofxodwlrq ri d jhqhudo udqjh ri shqhwudwlrq U4 1 Iru wklv sxusrvh wkh htxdwlrq
U4 @ 6H35 lv dgrswhg +U4 lq çj@fp5 ,1 Wkh ?op wklfnqhvv W / iru hdfk hohphqw l
lq wkh ?op/ lv qrz hvwlpdwhg dffruglqj wr=
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l
np
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frqwulexwlrqv iurp erwk ?op dqg vxevwudwh1
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lqlwldo Wi dqg Wv uhvshfwlyho|/ zkhuh Wv uhsuhvhqw wkh ?op wklfnqhvv edvhg xsrq wkh
vxevwudwh hohphqw phdvxuhphqw1 Lq fdvh wkh vdph hohphqw rffxuv lq erwk ?op dqg
vxevwudwh/ iru h{dpsoh dq r{lgh od|hu/ Do5 R6 / rq Do/ wkh +frpelqhg, n0udwlr iru wklv
ryhuodsslqj hohphqw lv xvhg lq wkh ?uvw hvwlpdwh iru wkh ?op wklfnqhvv lq wkh ?op
77
* +ã}, prghoolqj xvhg iru wklq ?op dqdo|vlv 7151
hohphqw f|foh dffruglqj wr=
Wil @ 3=8 ü U4 ü
l
l
ý np
ne>v
l ý nl
ne>v
e>i
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Wkh qh{w vwhs lv wr fdofxodwh wkh frpelqhg n0udwlrv/ nfl / zlwk wkh wklq ?op
surfhgxuh dv lq wkh suhylrxv sdudjudsk xvlqj wkh ?uvw hvwlpdwlrqv ri wkh ?op wklfnqhvv1
Wklv lv grqh iru hdfk lqyroyhg hohphqw1 Wkh frpelqhg n0udwlrv uhsuhvhqw wkh frpelqhg
frqwulexwlrqv iru wkh hohphqw l iurp wkh ?op dqg wkh vxevwudwh1
Vxevhtxhqwo| qhz hvwlpdwlrqv iru Wi dqg Wv kdyh wr eh pdgh e| frpsdulvrq
l
ri wkh fdofxodwhg n0udwlrv/ nfl / wr wkh phdvxuhg n0udwlrv/ np
1 Iru wklv wkh iroorzlqj
htxdwlrqv duh xvhg=
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l
np
nfl
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Li ryhuods ri dq hohphqw rffxuv/ ehfdxvh lw rffxuv lq erwk ?op dqg vxevwudwh/ wkh qhz
hvwlpdwlrq edvhg rq wkdw hohphqw lv jlyhq e|=
l
Wi>qhz
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l
l
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l
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Doo qhz hvwlpdwlrqv ri Wil dqg Wvl / edvhg rq wkh lqsxw iurp hdfk hohphqw lq ?op
dqg vxevwudwh/ duh qrz dyhudjhg djdlq dqg wkh surfhgxuh lv uhshdwhg1 Wkh lwhudwlyh
surfhvv lv vwrsshg rqfh wkh qhzo| fdofxodwhg ydoxhv iru W / gl>hu ohvv wkdq 314 (
iurp wkh suhylrxv rqhv/ ru zkhqhyhu qr frqyhujhqfh lv dfklhyhg zlwklq d uhdvrqdeoh
qxpehu ri f|fohv1 Xvxdoo| vxfk fdvhv lqglfdwh lqdghtxdwh phdvxulqj frqglwlrqv zkhuh/
iru h{dpsoh/ d wrr orz dffhohudwlrq yrowdjh lv xvhg dqg wkh * +ã}, fxuyh grhv qrw uhdfk
vx!flhqwo| lqwr wkh vxevwudwh1
71516
Ilop ri xqnqrzq wklfnqhvv dqg frpsrvlwlrq/ xqvxssru0
whg ru rq nqrzq vxevwudwh
Lq uhdolw|/ riwhq erwk wkh wklfnqhvv dqg wkh frpsrvlwlrq ri d ?op duh xqnqrzq1 Lw lv
reylrxv wkdw iru vxfk fdvhv d grxeoh lwhudwlyh surfhgxuh kdv wr eh xvhg1 Iru uhdvrqv
ri fodulw| zh zloo olplw wkh glvfxvvlrq wr wkh vlwxdwlrq zkhuh wkhuh lv qr ryhuods ri
hohphqwv lq ?op dqg vxevwudwh1
78
* +ã}, prghoolqj xvhg iru wklq ?op dqdo|vlv 7151
Dw wkh vwduw ri wkh grxeoh lwhudwlyh surfhgxuh/ wkh phdvxuhg n0udwlrv duh xvhg
dv gluhfw lqsxw iru wkh ?uvw hvwlpdwlrqv ri wkh zhljkw iudfwlrqv/ {l / ri wkh hohphqwv
suhvhqw1 Wkh zhljkw iudfwlrqv duh qrupdolvhg wr |lhog d wrwdo ri 433 (1 Qrz/ iru wklv
vwduwlqj frpsrvlwlrq doo fdofxodwlrqv ghvfulehg lq wkh suhylrxv vhfwlrq duh shuiruphg=
Wkh wklfnqhvv hvwlpdwlrqv duh pdgh dqg wkh hplwwhg lqwhqvlwlhv iru doo hohphqwv duh
fdofxodwhg1 Wkh uhvxowlqj fdofxodwhg n0udwlrv duh frpsduhg wr wkh phdvxuhg rqhv dqg
qhz hvwlpdwlrqv duh pdgh dffruglqj wr htxdwlrqv 71: dqg 71;1 Dw wklv srlqw dovr
qhz hvwlpdwlrqv iru wkh zhljkw iudfwlrqv duh pdgh vlpxowdqhrxvo|/ xvlqj wkh iroorzlqj
h{suhvvlrq=
{lqhz @ {lrog ü
l
np
nfl
+7143,
Diwhu hdfk f|foh lw lv fkhfnhg zkhwkhu frqyhujhqfh kdv ehhq rewdlqhg iru erwk ?op
wklfnqhvv dqg wkh zhljkw iudfwlrqv1 Wkh hqg ri wkh surfhgxuh lv qrz gh?qhg e| wzr
fulwhuld/ rqh iru wkh wklfnqhvv/ zklfk vkrxog ydu| ohvv wkdq 314 ( dqg d vhfrqg rqh iru
wkh zhljkw iudfwlrqv zkhuh wkh ghyldwlrq vkrxog eh zlwklq d fhuwdlq olplw/ vd| 313336
devroxwh1 Qrwh wkdw erwk fulwhuld vkrxog eh pdwfkhg dqg wkdw wkh olplwv iru wkhvh
fulwhuld fdq eh vhw dw fkrlfh1 Wkh ydoxhv xvhg khuh duh wdnhq iurp Edvwlq ^7`1
71517
Rwkhu/ pruh frpsoh{ fdvhv
Lw lv dovr srvvleoh wr vroyh pruh frpsoh{ vlwxdwlrqv zlwk wkh wklq ?op surjudp1 Hyhq
dq h{whqvlrq wr pxowlsoh od|huv dqg lq0ghswk sur?olqj kdv ehhq pdgh e| Edvwlq1 Lq
sulqflsoh/ lq0ghswk sur?olqj fdq eh grqh e| ydu|lqj wkh dffhohudwlrq yrowdjh/ dqg wkxv
wkh shqhwudwlrq ghswk1 Pruh lqirupdwlrq derxw pxowlod|hu surfhvvlqj fdq eh irxqg
lq ^7` dqg ^9`1
Li dq hohphqw lv suhvhqw lv erwk ?op dqg vxevwudwh/ hlwkhu wkh ?op wklfnqhvv
kdv wr eh vxssolhg/ ru/ rqh fdq xvh lqirupdwlrq rq wkh vwrlfklrphwu|1 Wkh odwwhu
fdvh lv riwhq xvhg iru fdofxodwlrq ri r{lghv/ zkhuh vwrlfklrphwu| lv zhoo0nqrzq1 Lw lv
dovr srvvleoh wr fdofxodwh wkh frpsrvlwlrq ri dq xqnqrzq vxevwudwh exulhg ehorz d
wklq od|hu ri nqrzq frpsrvlwlrq/ exw xqnqrzq wklfnqhvv1 Wklv lv yhu| xvhixo lq exon
phdvxuhphqwv zkhuh gholehudwho| d wklq ?op +xvxdoo| fduerq, kdv ehhq ghsrvlwhg rq
wkh vshflphq/ dqg fdq dovr eh xvhg wr wdnh lqwr dffrxqw vxuidfh od|huv olnh qdwlyh
r{lgh vnlqv1
Pruh lpsruw wr wklv wkhvlv/ krzhyhu/ lv wkh xvh ri wkh wklq ?op surfhgxuh
lq fdvh ri dq xqvxssruwhg wklq ?op1 Lq vxfk fdvh wkh dssur{lpdwlrq lv edvhg rq
wkh dvvxpswlrq wkdw wkh vxevwudwh frxog eh uhjdughg wr kdyh dq dwrplf qxpehu
dssurdfklqj }hur1 Wklv lv lqghhg wkh olqh ri uhdvrqlqj iroorzhg lq wklv wkhvlv1 Pruh
derxw wklv phwkrg zloo eh ghvfulehg lq wkh qh{w fkdswhu1
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Eleolrjudsk|
^4` J1 I1 Edvwlq dqg K1 M1 P1 Khlmoljhuv1 Txdqwlwdwlyh hohfwurq sureh plfurdqdo|vlv/
d qhz dssolfdwlrq= Wklq ?op dqdo|vlv1 Dwe Phwdooxujlh/ 6=:58:;/ +4<<3,1
^5` J1I1 Edvwlq/ M1P1 Glmnvwud/ K1M1P1 Khlmoljhuv/ dqg G1 Nohsshu1 Txdqwlwdwlyh
hohfwurq sureh plfurdqdo|vlv ri pxowl0od|hu vwuxfwxuhv1 Plnurfklplfd Dfwd ^Vxsso1`/
45=<68<:/ +4<<5,1
^6` J1I1 Edvwlq dqg K1M1P1 Khlmooljhuv1 Txdqwlwdwlyh HSPD ri wkh xowud0oljkw hohphqwv
erurq wkurxjk r{|jhq1 Plnurfklplfd Dfwd ^Vxsso1`/ 45=4<869/ +4<<5,1
^7` J1 I1 Edvwlq/ M1 P1 Glmnvwud/ K1 M1 P1 Khlmoljhuv/ dqg G1 Nohsshu1 Lq0ghswk sur?olqj
zlwk wkh hohfwurq sureh plfurdqdo|}hu1 Plfurehdp Dqdo|vlv/ 5=5<876/ +4<<6,1
^8` J1 I1 Edvwlq/ M1 P1 Glmnvwud/ dqg K1 M1 P1 Khlmoljhuv1 SUR]D<9= Dq lpsuryhg
pdwul{ fruuhfwlrq surjudp iru hohfwurq sureh plfurdqdo|vlv/ edvhg rq d grxeoh
jdxvvldq * +ã}, dssurdfk1 [0ud| Vshfwurp1/ 5:=6843/ +4<<;,1
^9` J1I1 Edvwlq dqg K1M1P1 Khlmoljhuv1 D v|vwhpdwlf gdwdedvh ri wklq ?op phdvxuh0
phqwv e| HSPD/ sduw L= Doxplqlxp ?opv1 Vxeplwwhg iru sxeolfdwlrq/ +4<<<,1
^:` M1O1 Srxfkrx dqg I1 Slfkrlu1 Xq qrxyhdx prgëoh gh fdofxo srxu od plfurdqdo|vh
txdqwlwdwlyh sdu vshfwurphwulh gh ud|rq {/ sduwlh LL= Dssolfdwlrq ã o*Dqdo|vh
g*Ìfkdqwloorqv kìwhurjëqh hq surirqghxu1 Uìfk1 Dìurvsdw1/ 8=67<869:/ +4<;7,1
^;` M1O1 Srxfkrx dqg I1 Slfkrlu1 Txdqwlwdwlyh dqdo|vlv ri krprjhqhrxv ru vwudwl?hg
plfuryroxphv dsso|lqj wkh prgho %SDS%1 Lq= N1I1M1 Khlqulfk dqg G1H1 Qhzexu|/
hglwruv/ Hohfwurq Sureh Txdqwlwdwlrq/ ss1 648:81 Sohqxp Suhvv/ Qhz \run dqg
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ri wklv lv xvhg iru dqdo|vlv ri exon vdpsohv zlwk vrph h{whqvlrqv wr wklq ?op phd0
vxuhphqwv ghyhorshg iru wkh HSPD1 Wklv phdqv lpsolflwo|/ wkdw wkh prghov zhuh
ghyhorshg iru HSPD dqg VHP frqglwlrqv= dffhohudwlrq yrowdjhv udqjlqj iurp dv orz
dv ÷ 5 nY xs wr d pd{lpxp ri 73 nY/ zlwk dq rffdvlrqdo phdvxuhphqw dw 83 nY1
Vlpxowdqhrxv zlwk wkh ghyhorsphqw ri wkhvh pdwul{ fruuhfwlrq surfhgxuhv iru
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o|vlv Gdwd iru [0Ud| Devruswlrq H>hfwv/ lq= Hohfwurq Plfurvfrs| dqg Dqdo|vlv1
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ri vdpsohv zlwk rqh nqrzq surshuw|1 yroxph 46/ ss1 9368937/ +4<<7,1
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64=::4/ +4<:8,1
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91514
Sureohpv dqg wkhlu vroxwlrqv
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wkh h{flwdwlrq yroxph dqg wkh * +ã}, fxuyh fkdqjh zkhq wkh vshflphq lv wlowhg zlwk
uhvshfw wr wkh hohfwurq ehdp/ wklv zdv qrw yhu| frqyhqlhqw iru wkh uhvhdufk rq wkh
qhz dqdo|vlv phwkrg/ ehfdxvh lw dgghg d qxpehu ri frpsolfdwlrqv wr vwduw zlwk1 Zh
zdqwhg wr nhhs wkh prghoolqj dv vwudljkwiruzdug dv srvvleoh1 Wkhuhiruh wkh ghwhfwru
zdv uhpryhg dqg uhexlow wr ?w wkh 73ï wlowhg sruw dydlodeoh rq wkh froxpq ri wkh I[
53331 Wkh fu|vwdo lq wkh ghwhfwru lv dovr wlowhg/ 63ï zlwk uhvshfw wr wkh ghwhfwru qr}}oh/
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91515
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wlphv odujhu wkdq wkdw lq d wklq WHP vshflphq/ wkh xvh ri exon vwdqgdugv zlwk vlplodu
ehdp fxuuhqwv dv xvhg rq d wklq WHP vshflphq/ zrxog ohdg wr kxjh [0ud| frxqw udwhv
iurp wkh exon vshflphqv/ fkrnlqj wkh HGV ghwhfwru1 Wkhuhiruh d vroxwlrq kdg wr eh
irxqg wr uhgxfh wkh [0ud| |lhog lq d frqwuroodeoh zd|1 Wzr vroxwlrqv wr wklv sureohp
frxog eh frqvlghuhg=
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wkh ghwhfwru lq fdvh ri d phdvxuhphqw rq d exon vdpsoh1 Wklv frxog eh uhdolvhg
e|/ iru h{dpsoh/ xvlqj d frqwuroodeoh dshuwxuh/ ru e| prylqj wkh ghwhfwru ixuwkhu
dzd| iurp wkh vshflphq1
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frqwuroodeoh dshuwxuh lv d srvvleoh vroxwlrq/ krzhyhu/ wkhuh lv olwwoh vsdfh wr prxqw
vxfk dq dshuwxuh ehwzhhq wkh fu|vwdo lq wkh HGV dqg wkh fduerq froolpdwru lq wkh
WHP1
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ehdp fxuuhqw uhtxluhv= 4, wkh kdugzduh rq wkh plfurvfrsh wr fkdqjh wkh ehdp fxuuhqw/
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91516
Ehdp fxuuhqw dqg vwdelolw|
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ohqv dshuwxuh/ ru fkdqjlqj wkh vsrwvl}h dqg2ru e| fkdqjlqj wkh eldv vhwwlqjv1 Wkh Mhro
5333 I[ kdv 6 vsrwvl}hv dydlodeoh lq VWHP prgh +V/P dqg O +dqg I prgh lq Orz
pdjql?fdwlrq,, dqg kdv 8 uhsodfhdeoh frqghqvhu ohqv dshuwxuhv1 Wkh dshuwxuhv zhuh
fkrvhq lq d odujh udqjh wr jhw d odujh ehdp fxuuhqw udqjh1 Vshfldo vpdoo dshuwxuhv/
vl}h 8 dqg 43 çp/ zhuh sxufkdvhg lq rughu wr xvh yhu| vpdoo ehdp fxuuhqwv qhhghg
iru phdvxuhphqwv rq wkh exon vwdqgdugv1 Wkh odujhu dshuwxuhv xvhg zhuh vwdqgdug
Mhro dshuwxuhv +53/ 453 dqg 533 çp,1 Wrjhwkhu zlwk wkh dydlodeoh vsrwvl}hv/ zklfk
surylgh urxjko| d idfwru 43 gl>huhqfh lq ehdp fxuuhqw/ wkh wrwdo ehdp fxuuhqw udqjh
fryhuv pruh wkdq 7 ghfdghv1 Wkh eldv vhwwlqj fdq eh xvhg iru ?qh dgmxvwphqwv ri wkh
ehdp fxuuhqw1 D iuhh ohqv frqwuro +dydlodeoh iurp Mhro, zrxog r>hu d frqwlqxrxv ehdp
fxuuhqw frqwuro1 Xqiruwxqdwho|/ wklv zdv qrw dw rxu glvsrvdo1
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dqdo|vlv/ zklfk fdq wdnh xs wr vhyhudo plqxwhv1 Xqiruwxqdwho|/ zh zhuh qrw deoh wr
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zlwk wkh lqlwldoo| xvhg FhE9 ?odphqw1 D vwdeoh ehdp fxuuhqw frxog eh rewdlqhg zlwk
d Wxqjvwhq ?odphqw/ dqg wkhuhiruh prvw h{shulphqwv ghvfulehg lq wklv wkhvlv zhuh
shuiruphg zlwk wkh Wxqjvwhq ?odphqw1 Diwhu d orqj vhdufk/ lq frrshudwlrq zlwk wkh
:6
Fkdswhu 91 H{shulphqwdo surfhgxuhv
vhuylfh hqjlqhhuv ri Mhro/ wkh ruljlq ri sureohp zdv hyhqwxdoo| irxqg wr eh lq wkh
frqwdfwv lq wkh Kljk Yrowdjh wdqn/ dqg vroyhg1
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jdyh ulvh wr ehdp lqvwdelolwlhv1 Sduw ri wkh ehdp lqvwdelolwlhv zhuh fdxvhg e| edg
frqwdfwv ehwzhhq ?odphqw fu|vwdo +lq fdvh ri d FhE9 ru OdE9 ?odphqw, dqg prxqwlqj
zluhv/ fdxvhg e| vpdoo fudfnv lq wkh fu|vwdo prxqw dqg wkh fu|vwdo lwvhoi1 Wkhvh fudfnv
rffxuuhg dv d uhvxow ri wrr idvw khdwlqj ri wkh ?odphqw1 Dq dxwrpdwlf ?odphqw khdwlqj
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ri wkh ?odphqw1
91517
HGV vwdelolw|
Gxulqj wkh phdvxuhphqwv/ wkh HGV sdudphwhuv zhuh nhsw dv vwdeoh dv srvvleoh= Vlqfh
n0udwlrv duh phdvxuhg/ prvw sdudphwhuv zloo fdqfho rxw lq wkh udwlr ehfdxvh wkh|
lq xhqfh wkh phdvxuhphqw rq erwk vwdqgdug dqg vshflphq +vdph [0ud| hqhuj| dqg
olqh, lq wkh vdph zd|1
Lq rughu wr nhhs wkh glvwdqfh ehwzhhq ghwhfwru fu|vwdo dqg vshflphq frqvwdqw/
dqg wkhuhiruh nhhslqj wkh froohfwlrq0 ru vrolg0dqjoh wkh vdph/ wkh ]0srvlwlrq ri wkh
vshflphq vkrxog eh wkh vdph dw hyhu| phdvxuhphqw1 Krzhyhu/ iru vshflphq dqg
vwdqgdugv/ gl>huhqw kroghuv duh xvhg1 Ixuwkhupruh/ qrw doo exon vwdqgdugv kdyh wkh
vdph khljkw1 Wr nhhs wkh vxuidfh ri wkh vshflphq ru vwdqgdug lq wkh vdph ]0srvlwlrq
hyhu| wlph/ wkh vxuidfh zdv doljqhg wr wkh hxfhqwulf srvlwlrq ri wkh vwdjh= Wklv lv d
phfkdqlfdoo| ?{hg srvlwlrq/ frpsohwho| lqghshqghqw ri plfurvfrsh vhwwlqjv ru vdpsoh1
916
Qhz ehdp fxuuhqw phwhu
Dv phqwlrqhg ehiruh/ d Sureh Fxuuhqw Ghwhfwru +SFG, zdv qhhghg lq rughu wr fkhfn
ehdp fxuuhqw vwdelolw| dqg wr phdvxuh wkh h{dfw ehdp fxuuhqw/ vr wkh [0ud| lqwhqvlwlhv
frxog eh h{suhvvhg lq fsv2qDv1 Wr ghvljq d uholdeoh SFG/ vrph h{shulphqwv zhuh
shuiruphg lq zklfk gl>huhqw rswlrqv zhuh whvwhg1 Wkh uhvxowv ri wkhvh h{shulphqwv
duh ghvfulehg lq wkh qh{w fkdswhu1 Qh{w/ dq ryhuylhz lv jlyhq ri wkh SFG ghvljq
frqvlghudwlrqv1
91614
SFG ghvljq
Wkh Jdwdq orz0edfnjurxqg kroghu/ zklfk zh kdg dw rxu glvsrvdo/ zdv surylghg zlwk
d Idudgd| fxs zlwk dq hohfwulfdo frqqhfwlrq wr d soxj dw wkh hqg ri wkh vshflphq
kroghu1 Lqvshfwlrq ri wklv Idudgd| fxs vkrzhg/ krzhyhu/ wkdw lw zdv idu iurp shuihfw
dqg glg qrw |lhog dffxudwh hqrxjk ehdp fxuuhqw phdvxuhphqwv1 Wkh soxj surylghg
:7
Qhz ehdp fxuuhqw phwhu 9161
iru wkh hohfwulfdo frqqhfwlrq wr d fxuuhqw phwhu fdxvhg qrlvh/ srru frqwdfw dqg d odujh
yrowdjh r>vhw fdxvhg e| hohfwurfkhplfdoo| lqgxfhg yrowdjhv1 Zh uhsodfhg wkh soxj e|
d orz qrlvh wuld{ldo soxj1 Shukdsv wkh odujhvw sureohp ri doo zdv wkdw wkh Idudgd| fxs
rq wkh kroghu lv lqfrqyhqlhqw vlqfh lw lv doprvw lpsrvvleoh wr uhwxuq wr wkh dqdo|vlv
orfdwlrq diwhu hdfk ehdp fxuuhqw phdvxuhphqw1
Khqfh/ d pruh vxlwdeoh SFG kdg wr eh ghvljqhg1 Wkh SFG kdg wr eh lqvhuwdeoh
lqwr wkh ehdp/ lqghshqghqwo| ri wkh vshflphq kroghu= Iru wklv sxusrvh wkh phfkdqlfdo
sduw ri d edfnvfdwwhuhg hohfwurq ghwhfwru 0 pdgh e| Mhro Qhwkhuodqgv 0 zdv xvhg1 Wkh
wls ri wklv ghwhfwru/ zklfk lv qrupdoo| lqvhuwhg mxvw deryh wkh vshflphq dqg krogv wkh
5 edfnvfdwwhuhg hohfwurq ghwhfwru hohphqwv/ zdv uhsodfhg e| dq hohfwulfdoo| lvrodwhg
wls1 D vklhoghg hohfwulfdo frqqhfwlrq zdv pdgh ehwzhhq wkh wls dqg d vshfldo Nhlwkoh|
orz vljqdo soxj/ prxqwhg dw wkh hqg ri wkh EH ghwhfwru phfkdqlfdo sduw +?jxuh 915
vkrzv wkh EHL sduw/ zlwk qhz wls dqg soxj,1
Iljxuh 9151 Sureh Fxuuhqw Ghwhfwru +SFG, zlwk prxqwlqj dqjh lq wkh plggoh1 Wkh
phfkdqlfdo sduw lv edvhg rq d EHL ghwhfwru/ zlwk d gl>huhqw wls +ohiw, dqg wuld{ldo orz
qrlvh frqqhfwru rq wkh uhdu vlgh +uljkw,1
Wkh ?uvw lghd zdv wr xvh d Idudgd| fxs rq wkh lqvhuwdeoh SFG wls/ lq rughu wr
fdswxuh 433( ri wkh ehdp fxuuhqw1 Krzhyhu/ wzr sureohpv durvh1 Iluvwo|/ wkh yhuwlfdo
vsdfh dydlodeoh zdv rqo| 518 pp lq rughu wr pdlqwdlq hqrxjk vdihw| glvwdqfh wr wkh
fduerq dshuwxuh dqg wr wkh vshflphq lq wlowhg srvlwlrq1 Wklv uhtxluhg d volp Idudgd|
fxs ghvljq1 Vhfrqgo|/ lq rughu wr fdswxuh wkh frpsohwh ehdp/ dw dq| ehdp srvlwlrq/
wkh ehdp hqwudqfh kroh ri wkh fkdpehu0olnh Idudgd| fxs kdg wr eh txlwh odujh1 Wkhvh
uhtxluhphqwv zrxog ohdg wr wkh frqvwuxfwlrq ri d Idudgd| fxs zklfk lv qrw deoh wr
fdswxuh 433( ri wkh ehdp fxuuhqw/ pdnlqj lw xquholdeoh1
Wkh dowhuqdwlyh zdv wr gurs wkh ghpdqg ri fdswxulqj 433( ri wkh ehdp fxuuhqw/
dqg wr eh vdwlv?hg zlwk d uholdeoh phdvxuhphqw ri d ?{hg shufhqwdjh ri wkh ehdp
fxuuhqw1 Wklv zdv grqh e| xvlqj d srolvkhg dqg fduerq frdwhg dw SFG wls/ vwloo
wklfn hqrxjk wr vwrs grzq wkh lqflghqw hohfwurqv frpsohwho|1 Dowkrxjk vxfk d dw
:8
Fkdswhu 91 H{shulphqwdo surfhgxuhv
wls zloo hplw vhfrqgdu| dqg edfnvfdwwhuhg hohfwurqv/ wkh fxuuhqwv lqyroyhg zlwk wklv
duh d ?{hg shufhqwdjh ri wkh wrwdo ehdp fxuuhqw +dw rqh vshfl?f dffhohudwlrq yrowdjh,1
Vlqfh wkh lqsxw sdudphwhuv iru txdqwlwdwlyh dqdo|vlv duh n0udwlrv/ wkh shufhqwdjh ri
wkh dfwxdo ehdp fxuuhqw fdqfhov rxw lq wkh udwlr +htxdwlrq 916,/ dqg wkhuhiruh lv qrw
uhohydqw1
Wkh ?qdo ghvljq ri wkh wls zdv dq doxplqlxp wls zlwk d srolvkhg lqvhuw ri
ylwuhrxv fduerq dw wkh hohfwurq ehdp fdswxulqj srvlwlrq dqg d vsxwwhuhg fduerq
frdwlqj ?qlvk rq wrs1 Wkh xvh ri fduerq/ ehlqj d oljkw hohphqw/ uhvxowv lq orz dprxqwv
ri edfnvfdwwhuhg dqg vhfrqgdu| hohfwurqv1 Wkhuhiruh/ pruh wkdq <3( ri wkh wrwdo ehdp
fxuuhqw fdq eh fdswxuhg1
91615
Fxuuhqw phwhu
Iru wkh dfwxdo fxuuhqw phdvxuhphqw/ d 9845 Nhlwkoh| hohfwurphwhu zdv xvhg dv d
fxuuhqw phwhu1 Zlwk vrph dgylfh iurp Nhlwkoh| d uholdeoh/ orz qrlvh/ orz fxuuhqw
phdvxulqj ghylfh zdv vhwxs1 Orz qrlvh wuld{ldo/ grxeoh vklhoghg frqqhfwruv +FV0964,
dqg orz qrlvh fdeohv +VF055, zhuh xvhg wr frqqhfw wkh phwhu wr wkh SFG1 Wr suhyhqw
jurxqg orrs fxuuhqwv/ d fhqwudo vklhog jurxqglqj srlqw zdv fkrvhq dw wkh fxuuhqw phwhu
lqsxw1 Wkh 7 45 gljlw 9845 hohfwurphwhu kdv fxuuhqw phwhulqj udqjhv iurp 53 pD +÷
3148( uhdglqj . 4 çD, wr 5 sD +÷ 419( ugj1 . 919 iD,/ doorzlqj dffxudwh fxuuhqw
phwhulqj grzq wr ehdp fxuuhqwv ri 314 sD1
917
Fkrlfh ri v|vwhpv xvhg iru whvwlqj wkh ) E45ä wklq
?op prgho1
Lq rughu wr whvw wkh qhz dqdo|vlv phwkrg/ vshflphqv zlwk erwk uhodwlyho| kljk dqg
uhodwlyho| orz dwrplf qxpehu hohphqwv zhuh qhhghg1 Lq vxfk vshflphqv wkh fkdudf0
whulvwlf [0ud|v iurp wkh oljkw hohphqw duh kljko| devruehg zkloh wkh [0ud|v iurp wkh
kljkhu dwrplf qxpehu hohphqwv duh doprvw devruswlrq iuhh1 H{dpsohv ri vxfk v|vwhpv
duh Doxplqlxp lq frpelqdwlrq zlwk hohphqwv olnh Wlwdqlxp/ Qlfnho ru ]lufrqlxp1
D ixuwkhu uhtxluhphqw ri wkh whvw pdwhuldov lv wkdw wkh| fdq eh suhsduhg zlwk
h{dfw/ zhoo0nqrzq dqg krprjhqhrxv frpsrvlwlrqv dqg wkh| vkrxog eh vwdeoh hqrxjk
wr suhsduh wklq WHP vshflphqv/ hlwkhu e| phfkdqlfdo julqglqj frpelqhg zlwk lrq
ehdp ploolqj ru e| hohfwurfkhplfdo wklqqlqj dqg srolvklqj1
Wdnlqj wkhvh uhvwulfwlrqv lqwr dffrxqw/ wkh ?uvw whvwlqj wr ?qg rxw zkhwkhu wkh
* +ã}, *SUR]D* surjudp zrunv zhoo xs wr 533 nY/ zdv grqh xvlqj exon vwdqgdugv
dqg exon vshflphqv zlwk nqrzq frpsrvlwlrq> Dujrq duf phowhg elqdu| frpsrxqgv/
DoQl/ DoWl/ Do6 Wl/ Do6 Qe/ Do6 ]u7 / Do5 ]u/ DoDx5 zhuh xvhg/ wrjhwkhu zlwk sxuh Do/
:9
Fkrlfh ri v|vwhpv xvhg iru whvwlqj wkh * +ã}, wklq ?op prgho1 9171
Wdeoh 9141 Frpsrvlwlrq ri elqdu| frpsrxqgv xvhg iru vshflphqv dv phdvxuhg zlwk
HSPD1 Wkh suhvhqwhg ghyldwlrqv lqglfdwh wkh <8 ( frq?ghqfh udqjh dqg wkh ydoxhv
ehwzhhq eudfnhwv duh wkh frpsrvlwlrqv dffruglqj wr vwrlfklrphwu|1,
Frpsrxqg
DoQl
DoWl
Do6 Wl
Do6 Qe
Do6 ]u7
Do5 ]u
DoDx5
Dwrp ( Do
7;1< ÷ 316 +8313,
7<17 ÷ 31: +8313,
:61< ÷ 319 +:813,
:61; ÷ 315 +:813,
7516 ÷ 316 +751<,
971; ÷ 314 +991:,
651; ÷ 315 +6616,
Dwrp( P
8414 ÷ 316 +8313,
8319 ÷ 31: +8313,
5914 ÷ 319 +5813,
5915 ÷ 315 +5813,
8:1: ÷ 316 +8:14,
6815 ÷ 314 +6616,
9:15 ÷ 315 +991:,
Ql/ Wl/ Qe/ ]u/ dqg Dx vwdqgdugv1 Vxevhtxhqwo|/ wklq WHP vdpsohv zhuh suhsduhg
e| glpsoh julqglqj dqg lrq ploolqj ri wkh DoWl/ Do6 Wl/ DoQl dqg wkh Do6 ]u7 wr whvw
wklq ?op dqdo|vlv1
91714
Exon vshflphq frpsrvlwlrqv
Wkh frpsrvlwlrq ri doo vdpsohv zdv fkhfnhg lq rxu Mhro ;933 HSPD wdnlqj 63 dqdo|vlv
srlqwv +53 nY,1 Wkh dyhudjh n0udwlrv zhuh xvhg wr fdofxodwh wkh frpsrvlwlrqv xvlqj
wkh qhz *SUR]D<9* surjudp zlwk wkh grxeoh Jdxvvldq prgho1 Wkh Do6 Wl/ Do6 Qe/
Do6 ]u7 / dqg wkh Do5 ]u duh olqh frpsrxqgv/ zkloh wkh DoQl/ DoWl/ dqg wkh DoDx5 duh
qrw ^6`1 Wkh Do6 ]u7 vshflphq zdv irxqg wr frqwdlq vpdoo duhdv ri Do5 ]u6 skdvh1 Dq
ryhuylhz ri wkh HPSD dqdo|vlv uhvxowv lv vkrzq lq Wdeoh 9141
Wkh uhvxowv ri wkhvh HSPD dqdo|vhv duh frpsduhg zlwk wkh WHP dqdo|vlv
uhvxowv rewdlqhg odwhu dqg zklfk duh wr eh suhvhqwhg lq wkh qh{w fkdswhu1
91715
Xvhg suhsdudwlrq whfkqltxhv
Wkh vdpsohv dv zhoo dv wkh vwdqgdugv zhuh suhsduhg e| phfkdqlfdo julqglqj dqg
srolvklqj1 Hohfwurfkhplfdo srolvklqj zdv dssolhg iru wkh ?uvw doxplqlxp vwdqgdugv
dqg iru dq DoQl vshflphq/ exw wkhvh vshflphqv zhuh glvfdughg vlqfh srolvklqj zdv
qrw shuihfw dqg wkh phwkrg frxog qrw eh dssolhg wr rewdlq d jrrg exon vwdqgdug lq
wkh fdvh ri doxplqlxp ehfdxvh wkh ?qdo vshflphq wklfnqhvv zdv wrr vpdoo1
Diwhu wklqqlqj wkh pdwhuldo grzq zlwk frduvh julqglqj sdshu wr volfhv ri derxw
4 pp wklfnqhvv/ 6 pp glvfv zhuh fxw rxw e| xowudvrqlf guloolqj/ +ru lq fdvh ri pruh
eulwwoh pdwhuldo/ vdzlqj vtxduhv dqg vlpso| julqglqj wkhp wr 6 pp urxqg glvfv e|
kdqg,1 Vxevhtxhqwo|/ lqlwldo wklqqlqj ri doo pdwhuldov zdv grqh e| julqglqj rq VlF
julqglqj sdshu xvlqj julw vl}hv +Hxurshdq, 933/ 4533/ 5833 dqg 73331 Ilqdoo| wkh
::
Fkdswhu 91 H{shulphqwdo surfhgxuhv
Wdeoh 9151 Srolvklqj phwkrgv xvhg iru sxuh hohphqwv vwdqgdugv dqg exon elqdu| frp0
srxqgv1
Pdwhuldo
Do
Wl2Ql2Fr
]u2Qe
Qe
DoWl2Do6 ]u7
DoQl2DoDx5
DoQe6
4
5
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Do +Kduwihow ) Fr1,
5
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K|gursdo ð
Do +Kduwihow ) Fr1,
srolvklqj djhqw
u
Kljk frqf1 Ilqdoð
+VlR5 ,
u
ð
Ilqdo +VlR5 ,
Zdwhu +dfwlyh srolvklqj glvf,
6çp/ 4çp Gldprqg lq Wulsoh
Ilqdo +VlR5 ,
4çp/ 3158çp Gldprqg
Zdwhu
u
ð
Vshfldo forwk iru VlR5 vroxwlrq/ sxufkdvhg iurp *Yrv hq ydq Hlmn Phwdooxujlh*
Ilqh orz sroh forwk/ sxufkdvhg iurp *Yrv hq ydq Hlmn Phwdooxujlh*
pdwhuldov zhuh srolvkhg e| wkh phdqv lqglfdwhg lq wdeoh 9151 Wkh ?qdo exon whvw
vshflphqv zhuh dssur{lpdwho| 318 pp wklfn zklfk lv lq doo fdvhv wklfn hqrxjk wr vwrs
grzq lqflghqw hohfwurqv ri 533 nY frpsohwho|1
Diwhu wkh exon phdvxuhphqwv kdg ehhq shuiruphg/ WHP wklq ?op vshflphqv
zhuh suhsduhg rxw ri exon vdpsohv1 Ixuwkhu wklqqlqj iurp rqh vlgh wr derxw 315 pp
zdv dssolhg/ iroorzhg e| glpsoh julqglqj xvlqj d Jdwdq glpsoh julqghu zlwk 53 pp
frsshu zkhhov zlwk 9 çp dqg 4 çp gldprqg lq rlo/ iroorzhg e| d ?qdo srolvk zlwk
u
srolvklqj zkhho dqg Do5 R6 ru VlR5 +Ilqdo ð
,/ grzq wr 43 0 53 çp wklfnqhvv lq wkh
fhqwuh ri wkh glpsoh1 Lrq ehdp ploolqj zdv xvhg wr rewdlq wkh ?qdo vshflphqv1
91716
Vwdqgdugv
Iluvwo|/ 6 pp exon vshflphqv iurp sxuh phwdov zhuh xvhg dv vwdqgdug pdwhuldov1
Wkh srolvkhg exon vdpsohv zhuh prxqwhg lq wkh Jdwdq orz edfnjurxqg kroghu1 Wklv
ghpdqghg fkdqjlqj lq dqg rxw wkh vshflphq kroghu iru hdfk qhz vwdqgdug1
Odwhu/ d vshfldo vohgjh0w|sh plql kroghu zdv pdgh rxw ri kljk fduerq frqwhqw
hsr{| pdwhuldo +vhh Iljxuh 916,1 Wklv orz edfnjurxqg plql kroghu zlwk wkh vwdqgdugv
zdv wkhq prxqwhg lq d jhqhudo0sxusrvh Mhro exon kroghu +vhh Iljxuh 917,1 Wkh plql
kroghu zdv proghg iurp WHP hpehgglqj hsr{| uhvlq +845 sroduehg . PQD, zlwk
dssur{lpdwho| 6 yroxph sduwv fohdq +khdwhg dqg ydfxxp wuhdwhg, dfwlyh fduerq
+wklv lv ?qho| vwuxfwxuhg/ dpruskrxv fduerq zklfk zloo qrw vphdu gxulqj julqglqj
ru srolvklqj,1 Ilqdoo|/ wkh plql kroghu zdv jrog frdwhg rq wkh edfnvlgh wr dfklhyh
rswlpdo hohfwulfdo frqwdfw wr wkh exon kroghu1
Wkh vwdqgdugv wkhpvhoyhv zhuh 317 0 318 pp wklfn/ 418 { 6 pp zlgh vwulsv ri
srolvkhg pdwhuldo +fdq eh srolvkhg lq exon, zklfk fdq eh volg lqwr wkh plql kroghu1
:;
Vriwzduh 9181
Iljxuh 9161 Plql exon vwdqgdugv kroghu/ pdgh ri Fduerq ?oohg sro|phu/ wr eh prxqwhg
lqwr d jhqhudo exon vshflphq kroghu
Xvlqj wklv v|vwhp/ xs wr derxw 9 vwdqgdugv fdq eh srvlwlrqhg lq dq dvvhpeo| wr
irup d eorfn ri vwdqgdugv1 Vpdoohu exon sduwlfohv fdq eh hpehgghg lqwr wkh vdph
fduerq0frqwdlqlqj uhvlq wr vlploduo| vkdshg vwdqgdug vwulsv1 Wklv lv xvhixo zkhq wkh
exon pdwhuldo fdq qrw eh iruphg lqwr d 6 pp zlgh vwuls +h1j1 li rqo| vpdoo oxpsv
ru fu|vwdov duh dydlodeoh,1 Il{hg vwdqgdug eorfnv fdq eh pdgh xvlqj wkh vdph uhvlq
wr prxqw vhyhudo exon vwdqgdugv lqwr rqh eorfn1 Lw vkrxog eh qrwhg djdlq wkdw hdfk
vwdqgdug pxvw eh dw ohdvw 316 pp wklfn +iru Do lq d 533 nY plfurvfrsh, wr pdnh vxuh
wkdw wkh hohfwurqv duh frpsohwho| vwrsshg zlwklq wkh uhihuhqfh pdwhuldo1
918
Vriwzduh
Doo vshfwud duh dftxluhg xvlqj wkh Qrudq *PlfurT* surjudp1 Wkh shdn ?wwlqj lv dovr
shuiruphg lq PlfurT/ xvlqj xvhu gh?qhg uhihuhqfhv1 Wkh uhjlrqv ri lqwhuhvw +URL,
duh xvxdoo| wdnhq ryhu wkh hqwluh shdn +Nò . Nñ , zlwk vrph edfnjurxqg rq erwk
vlghv ri wkh shdn wr hqvxuh surshu shdn ?wwlqj1 Rffdvlrqdoo|/ d vlqjoh Nò 0olqh lv xvhg/
zkhq shdn vhsdudwlrq ehwzhhq Nò dqg Nñ lv odujh hqrxjk wr rewdlq dq dghtxdwh
edfnjurxqg ehwzhhq wkh wzr shdnv1
Olqhvfdqv/ w|slfdoo| frqvlvwlqj ri 63 srlqwv ryhu d udqjh ri 4 wr 53 çp/ duh
fduulhg rxw xvlqj wkh *DxwrT* frppdqg ri PlfurT/ ru e| d vlpsoh vfkhgxoh frqwuroolqj
dftxlvlwlrq dqg ehdp pryhphqw1 D vpdoo prgl?fdwlrq wr wkh PlfurT surjudp pdgh
lw srvvleoh wr vdyh hdfk vshfwuxp dftxluhg lq wkh olqhvfdqv/ vr doo surfhvvlqj frxog eh
grqh vhsdudwho| diwhuzdugv li qhhghg1 Doo vshfwud ri vwdqgdugv zhuh rewdlqhg xvlqj
pxowlsoh dqdo|vlv srlqwv ru lq vorz vfdq prgh/ wr plqlplvh huuruv gxh wr vxuidfh ghihfwv
:<
Fkdswhu 91 H{shulphqwdo surfhgxuhv
Iljxuh 9171 Vwdqgdugv eorfn prxqwhg lq wkh Mhro exon vshflphq kroghu1
ru vpdoo orfdo lqkrprjhqhlw|1
Wklq ?op * +ã}, fdofxodwlrqv ri frpsrvlwlrq dqg wklfnqhvv zhuh shuiruphg zlwk
wkh WID<<< surjudp ^7`^8`1 Wkh *SUR]D<9* surjudp ^9`zdv xvhg wr fdofxodwh fru0
uhfwlrq idfwruv qhhghg lq fdvh ri frpsrxqg exon vwdqgdugv1
919
Ilqdo vhwxs
Diwhu doo wkh prgl?fdwlrqv lq wkh vhwxs ri wkh WHP dqg lq wkh dqdo|vlv v|vwhp/ dv
ghvfulehg lq wklv fkdswhu wkh ?qdo vhwxs lv dv iroorzv=
î Mhro I[ 5333/ zlwk OdE9 ?odphqw zlwk dxwrpdwlf khdwlqj xqlw1 Frqghqvhu
dshuwxuhv 533/ 453/ 53/ 43 dqg 8 çp +314pp sodwlqxp, duh xvhg wr dfklhyh d
yhu| zlgh fxuuhqw udqjh1 Wkh 43 dqg 8 çp dshuwxuh duh rewdlqhg iurp Djdu/
wkh 453 dq 533 çp duh Mhro w|shv1 Dv phqwlrqhg/ wkh hduolhu phdvxuhphqwv zhuh
shuiruphg zlwk d Wxqjvwhq ?odphqw/ odwhu d OdE9 +Mhro, ?odphqw zdv xvhg1
î D Qrudq HGV zlwk Qruydu zlqgrz lv prxqwhg dw 73ï zlwk uhvshfw wr wkh
vshflphq sodqh/ xvlqj dq lqfolqhg fu|vwdo uhvxowlqj lq d :3ï wdnh0r> dqjoh dqg d
vkruw ghwhfwru0wr0vshflphq glvwdqfh1
î D Jdwdq orz edfnjurxqg vshflphq kroghu zlwk Ehu|oolxp lqvhuw lv xvhg iru
kroglqj wkh WHP vshflphqv/ dqg d Mhro exon vshflphq kroghu lv xvhg/ wr krog
;3
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zlwk rqo| rqh pdmru prgl?fdwlrq= wkh lqvwdoodwlrq ri wkh sureh fxuuhqw phwhu1 Zlwk
447
Uhfrpphqgdwlrq iru ixuwkhu uhvhdufk dqg lpsuryhphqwv wr wkh WHP vhwxs ;161
wkh rewdlqhg uhvxowv/ lw lv qrw srvvleoh wr uh?qh wkh *WID* surjudp/ wkh wklq ?op
yhuvlrq ri wkh *SUR]D surjudp1 Suredeo|/ wkh fdofxodwlrqv e| wkh surjudp duh
pruh dffxudwh wkdq zh fdq phdvxuh/ hyhq lq WHP frqglwlrqv1 Ehwwhu dqdo|vlv uhvxowv
frxog eh rewdlqhg e| hlwkhu grlqj pdq| pruh phdvxuhphqwv/ hqodujlqj wkh gdwdedvh
dqg khqfh/ uhgxflqj wkh vwdwlvwlfdo huuruv ru e| xvlqj d plfurvfrsh pruh vshfl?fdoo|
ghvljqhg dqg exlow iru dqdo|vlv sxusrvhv +iru h{dpsoh dv lqglfdwhg e| O|pdq +4<<7,
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Ixuwkhupruh/ lw vkrxog eh qrwhg wkdw lq wklq ?opv rwkhu sdudphwhuv/ vxfk dv
fu|vwdo rulhqwdwlrq/ fdq lq xhqfh wkh phdvxuhphqwv1 Zkhq lpsurylqj wkh vsdwldo
uhvroxwlrq hyhq ixuwkhu/ grzq wr wkh vxe0qdqrphwhu vfdoh +rqo| srvvleoh rq yhu| wklq
vshflphqv,/ wkh h{flwdwlrq yroxph ehfrphv vr vpdoo wkdw rqh fdqqrw dvvxph d krpr0
jhqhrxv glvwulexwlrq ri wkh hohphqwv dq| pruh/ dqg h>hfwv gxh wr wkh olplwhg qxpehu
ri dwrpv pd| ehfrph lpsruwdqw1 +vhh dovr ^9`,1
Frqfhuqlqj wkh hodvwlf vfdwwhulqj htxdwlrqv xvhg lq wkh prgho/ lw lv vrphwlphv
phqwlrqhg wkdw fruuhfwlrqv vkrxog eh pdgh iru uhodwlylvwlf h>hfwv ^:`1 D sursrvhg
phwkrg wr fruuhfw iru wkhvh h>hfwv/ lv e| lqfuhdvlqj wkh ydoxh ri wkh dffhohudwlrq
yrowdjh/ H3 1 Krzhyhu/ li rqh zrxog frqvlghu lqfrusrudwlqj vxfk uhodwlylvwlf fruuhfwlrq/
qrw rqo| wkh zdyhohqjwk +dqg wkhuh iru H3 , fkdqjhv/ exw pdq| pruh sdudphwhuv/ vxfk
dv wkh vshflphq wklfnqhvv zklfk lv vhhq e| wkh hohfwurq sdvvlqj wkurxjk1 Pruhryhu/
li dq| uhodwlylvwlf h>hfwv duh suhvhqw/ wkh| duh suredeo| douhdg| sduwldoo| hqforvhg e|
wkh pdq| sdudphwhuv lq wkh prgho1
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Uhfrpphqgdwlrq iru ixuwkhu uhvhdufk dqg lp0
suryhphqwv wr wkh WHP vhwxs
Dv phqwlrqhg lq wkh suhylrxv vhfwlrq/ pruh dffxudwh phdvxuhphqwv/ dqg2ru vlpso|
pdq| pruh phdvxuhphqwv/ duh qhhghg wr gr dq| lpsuryhphqwv wr wkh *SUR]D* dqg
wkh *WID<<<* surjudpv dqg wkh prghov rq zklfk wkh| duh edvhg1 Lq rughu wr gr vr/
d frxsoh ri vpdoo lpsuryhphqwv fdq vwloo eh pdgh wr wkh fxuuhqw vhwxs=
î Wkh SFG vkrxog eh dxwrpdwhg/ vlplodu wr dq HSPD1 Dq dxwrpdwhg SFG pdnhv
lw srvvleoh wr shuirup olqhvfdqv zlwk odujh dprxqw ri dqdo|vlv srlqwv dqg orqjhu
dftxlvlwlrq wlphv/ vlqfh wkh ehdp fxuuhqw fdq eh phdvxuhg ehiruh hyhu| srlqw
zlwkrxw lqwhuihuhqfh ri dq rshudwru1 Iru plfurvfrshv zlwk dxwrpdwhg vwdjh
frqwuro/ lw pljkw ph srvvleoh wr xvh d SFG rq wkh vshflphq kroghu ru rq wkh
vwdjh lwvhoi/ zkhq lqvwdoodwlrq ri d SFG ghylfh lv qrw srvvleoh rq wkh vshfl?f
plfurvfrsh/ vlqfh wkh vwdjh dxwrpdwlrq zloo doorz wkh xvhu wr uhwxuq dffxudwh wr
wkh dqdo|vlv srvlwlrq1
448
Uhfrpphqgdwlrq iru ixuwkhu uhvhdufk dqg lpsuryhphqwv wr wkh WHP vhwxs ;161
î Dowkrxjk wkh hohfwurphwhu xvhg iru wkh fxuuhqw phdvxuhphqw lv dffxudwh hqrxjk/
d vpdoo lpsuryhphqw fdq eh uhdolvhg lq vlwxdwlrqv zkhuh d yhu| orz ehdp fxuuhqw
lv qhhghg +vxfk dv kljk dwrplf qxpehu exon vwdqgdugv, e| xvlqj dq hyhq pruh
dffxudwh fxuuhqw phwhu/ vxfk dv wkh Nhlwkoh| 9751 D vhsdudwh vljqdo dpsol?hu
orfdwhg dw wkh SFG lwvhoi/ fdq uhgxfh wkh vljqdo qrlvh lqgxfhg e| wkh fdeolqj1
î Lqvwdoodwlrq ri d iuhh ohqv fxuuhqw frqwuro/ zloo doorz frqwlqxrxv yduldwlrq ri wkh
ehdp fxuuhqw1 E| ydu|lqj wkh ehdp fxuuhqw/ wkh HGV ghdg wlph frxog eh nhsw
frqvwdqw/ dyrlglqj frxqw udwh sureohpv1
î Wkh odujhvw lpsuryhphqw fdq suredeo| eh dfklhyhg e| lpsurylqj wkh HGV
+frxqwlqj, v|vwhp/ vlqfh orz frxqw udwhv dqg d olqhdu ehkdylrxu ri wkh HGV
dqg lwv sxovh0surfhvvlqj duh wkh wzr pdmru vrxufhv ri huuruv lq wkh rewdlqhg
dqdo|vlv gdwd1 Iru h{dpsoh/ d gljlwdo sxovh0surfhvvru lv deoh wr kdqgoh pxfk
kljkhu frxqw udwhv wkdq wkh fxuuhqw dqdorjxh v|vwhp1
Ixuwkhupruh/ whvwlqj ri wkh prgho frxog eh h{whqghg wr wlowhg vshflphqv dqg
wr xowud0oljkw hohphqwv1 Wkh odwwhu lv hvshfldoo| lqwhuhvwlqj/ vlqfh lq wkhvh fdvhv
dq dssursuldwh devruswlrq fruuhfwlrq ehfrphv kljko| lpsruwdqw1 Dowkrxjk lw
pd| vhhp vrphzkdw shfxoldu/ frpelqlqj wkh oljkw hohphqw fdvhv dqg wklq ?op
dqdo|vlv lq d WHP pd| eh srvvleoh= Diwhu doo/ prvw ri wkh hplwwhg udgldwlrq
lq fdvh ri d oljkw hohphqw exon vdpsoh ruljlqdwhv iurp wkh yhu| vxuidfh ri wkh
vshflphq/ zkloh doo wkh udgldwlrq jhqhudwhg ghhshu lq wkh vshflphq lv devruehg
dqg zloo qrw eh hplwwhg1 Lq rwkhu zrugv= wkh * +ã}, fxuyh ri wkh jhqhudwhg [0ud|
lqwhqvlw| uhdfkhv wr odujh ghswkv lq wkh vshflphq zkloh rqo| wkh udgldwlrq iurp
wkh yhu| ehjlqqlqj ri wkh fxuyh lv hplwwhg dqg fdq eh phdvxuhg e| wkh ghwhfwru1
Wkhuhiruh dqdo|vlv ri oljkw hohphqwv lq exon vshflphqv lv yhu| gl!fxow1 Lq wkh
wklq ?op dqdo|vlv fdvh/ wklv sureohp lv qrw suhvhqw vlqfh [0ud|v duh jhqhudwhg
lq wkh wklq ?op dqg prvw ri wkhp zloo dovr eh hplwwhg1
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H{whqvlrqv ri wkh qhz phwkrg
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frpsrvlwlrq dw dq| dffhohudwlrq yrowdjh1 Wkhuhiruh lw vkrxog eh srvvleoh wr rewdlq
dq dffxudwh vshflphq wklfnqhvv ydoxh ri d WHP vshflphq ri nqrzq frpsrvlwlrq/ e|
phdvxulqj pxowlsoh gdwd srlqwv dw ydu|lqj wklfnqhvv/ ru e| ydu|lqj wkh dffhohudwlrq
yrowdjhv/ zlwkrxw phdvxulqj wkh exon vwdqgdug1 Zlwk d surjudp urxwlqh wr shuirup
wkh qhfhvvdu| fdofxodwlrqv/ wklv zrxog h{whqg wkh phwkrg/ pdnlqj lw vxlwdeoh iru xvlqj
erwk exon dqg wklq ?op vwdqgdugv1
449
Eleolrjudsk|
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7 0 wkh udwlr whfkqltxh1 Lq= Surf1 Iliwk Hxurshdq Frqjuhvv rq Hohfwurq Plfurvfrs|/
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vshfwud1 Lq= S1H1 Uxvvhoo/ hglwru/ Plfurehdp Dqdo|vlv/ ss1 8348839/ Vdq Iudqflvfr/
+4<;<,1 Vdq Iudqflvfr Suhvv1
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lq wklq0vdpsoh txdqwlwdwlyh plfurdqdo|vlv1 Xowudplfurvfrs|/ 54=5:485:9/ +4<;:,1
^8` F1H1 O|pdq/ M1L1 Jrogvwhlq/ G1E1 Zlooldpv/ G1Z1 Dfnodqg/ V Yrq Kduudfk/ D1Z1
Qlfkroov/ dqg S1M1 Vwdwkdp1 Kljk0shuirupdqfh {0ud| ghwhfwlrq lq d qhz dqdo|wlfdo
hohfwurq plfurvfrsh1 Mrxuqdo ri Plfurvfrs|/ 4:9=;88<;/ +4<<7,1
^9` M1L1 Jrogvwhlq/ G1E1 Zlooldpv/ dqg J1 Fol>1 Txdqwlwdwlyh {0ud| dqdo|vlv1 Lq= G1F
Mr|/ D1G1 Urplj/ Mu1/ dqg M1L1 Jrovwhlq/ hglwruv/ Sulqflsohv ri Dqdo|wlfdo Hohfwurq
Plfurvfrs|/ fkdswhu 8/ ss1 488854;1 Sohqxp Suhvv/ Qhz \run dqg Orqgrq/ +4<;9,1
^:` M1L1 Jrogvwhlq/ M1O1 Frvwoh|/ J1Z1 Orulphu/ dqg V1M1E1 Uhhg1 Txdqwlwdwlyh {0ud|
dqdo|vlv lq wkh hohfwurq plfurvfrsh1 Vfdqqlqj Hohfwurq Plfurvfrs|/ L=6488657/
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flphqv qhhghg iru wkh dqdo|vlv1 Vrph ri wkh sureohpv lqyroyhg zlwk wkh txdqwl?fdwlrq
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dqg duh wkhuhiruh lq jhqhudo rqo| vxlwhg iru shuihfwo| suhsduhg xowud wklq vshflphqv dqg
qrw iru pruh gl!fxow vlwxdwlrqv vxfk dv oljkw hohphqw dqdo|vhv1 Lq frqwudvw wr txdq0
wlwdwlyh dqdo|vlv rq d WHP/ pxfk pruh uhdolvwlf dqg frpsoh{ fruuhfwlrq prghov duh
ghyhorshg iru dqdo|vlv zlwk dq Hohfwurq Sureh Plfur Dqdo|vhu +HSPD,1 Zh irxqg
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