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Chng 12

Lp bn gene
Vic lp bn gene l mt bc quan trng trong vic tm hiu,
chn on v iu tr cc bnh di truyn. Hin nay c trn 14.000 gene
trong s khong t 30.000 n 40.000 gene trong genome ca ngi
c xc nh v tr trn nhim sc th v ch khong 1.500 bnh do t
bin gene xc nh c t bin trn cc gene c hiu. Nh vy r
rng l vn cn rt nhiu vic phi lm tm hiu cc bin i xy ra trn
gene lm gy ra nhng bnh di truyn.
C hai cch lp bn gene chnh: (1) Lp bn di truyn
(genetic mapping) l phng php trong tn s hon v gia cc locus
trn NST qua gim phn c s dng nh gi khong cch gia cc
locus v (2) lp bn vt l (physical mapping) lin quan ti vic s
dng cc k thut phn t v di truyn t bo xc nh v tr vt l ca
cc gene trn NST.

I. Lp bn di truyn (genetic mapping)


1.Hin tng trao i cho (crossing over) gia cc gen lin kt
Trong gim phn gia cc NST tng ng i khi xy ra s trao
i cc on DNA trong k u ca ln phn bo I gi l trao i cho.
Mt NST c kch thc trung bnh s c t 1 n 2, 3 v tr trao i cho
trong gim phn qua c th lm xy ra s ti kt hp ca cc allele trn
cp NST tng ng.
2. Tn s hon v
S trao i cho xy ra gia cc locus nm xa nhau trn 1 NST
nhiu hn gia cc locus nm cnh nhau (hnh 2). Nh vy khong cch
gia 2 locus c th c tnh ton c bng cch nh gi tn s ti t
hp xy ra trong cc gia nh (hin tng hon v gene). Nu trong mt s
lng ln cc ln gim phn c nghin cu trn cc gia nh, cc allele
A v B tri qua ti t hp 5% ln th tn s hon v gia A v B s l
5% (0,05).
Khong cch di truyn gia 2 locus c o bng n v
centiMorgan (cM) tng nh ti Morgan, ngi pht hin ra hin
tng bt cho ca cc NST vo nm 1910. Mt cM tng ng vi 1%
tn s hon v, mi 1cM tng ng vi khong 1 triu bp (1Mb). Nu 2
locus lin kt cch nhau 50cM th c coi nh l khng lin kt, v trong
trng hp ny tn s hon v ca chng bng vi khi chng di truyn

phn ly c lp vi nhau.
3. Cch tnh tn s hon v
Tn s hon v c nh gi thng qua vic kho st s di truyn
cc gene trong ph h.
Hnh 1 l mt ph h ca bnh u x thn kinh type 1 (NF1) trong
cc thnh vin c phn loi theo mt RFLP c hai allele gi l 1F10 (k
hiu l 1 v 2), RFLP ny cng nm trn NST 17 vi gene NF1. Kiu gene
1F10 c ghi pha di mi c th trong ph h.
Kho st th h th nht v th h th hai cho thy gene NF1 phi
lin kt vi allele 1 ca 1F10 trn cng 1 NST trong gia nh ny, v c
th I-2 ng hp t v allele 2 (2,2) khng mc bnh. Ngi b mc bnh
(I-1) d hp t locus 1F10 (1,2) truyn mt NST cha c allele bnh
v allele1 ca 1F10 cho con gi (II-2).

Hnh 1: A, Mt ph h ca bnh NF1 trong mi thnh vin c phn loi


da trn tnh a hnh ca 1F10. Kiu gene ca locus marker gm 2 allele ny
c ghi di mi c th trongph h. Cc thnh vin trong ph h b mc bnh
c bi en. B, Hnh phng x t chp ca cc allele ca marker 1F10 trong
ph h ny.

S sp xp ca cc allele ny trn mi NST c gi l phase lin


kt (linkage phase). Khi bit v phase lin kt ri th kiu gene ca c

con gi II-2 khi s l N1/n2 (vi N l allele gy bnh NF1 v n l allele


bnh thng). Chng ca c ny (II- 1) khng mc bnh v ng hp t v
allele 2 locus 1F10 (2,2) do c kiu gene l n2/n2. Nhng a con
ca cuc hn nhn ny (th h III) nu mc bnh NF1 th s mang allele
bnh N i cng vi allele 1 ca locus 1F10 trong khi nhng a tr
khng mang bnh s mang allele 2 ca locus 1F10 i cng vi allele lnh
n.7 trong 8 a tr ca th h th ba cho thy iu ny hon ton ng.
Tuy nhin c mt trung hp xy ra hin tng ti kt hp
(ngi III-6). iu ny cho thy mt tn s hon v 1/8 hay 12,5%. Tn s
ny gia cc locus khng allele h tr cho gi thuyt v s lin kt gia
locus NF1 v 1F10. Nu y xut hin mt tn s ti t hp l 50% s
h tr cho gi thuyt l hai locus ny khng lin kt vi nhau tc l di
truyn phn ly c lp vi nhau.
Trong thc t mt mu ln hn gm nhiu gia nh s c s dng
m bo gi tr v mt thng k ca kt qu ny. Nh vy vic nh gi
tn s ti kt hp bng cch kho st s di truyn ca cc allele trong
nhiu gia nh v vic xc nh phase lin kt gip nghin cu v tr ca
cc gene trn NST v qua lp c bn gene.
4. Vai tr ca cc marker
Hin tng a hnh (polymorphism) trn locus 1F10 c s dng
theo di mt gene bnh no trong gia nh c gi l cc marker.
Chng c dng nh du NST c mang allele bnh. V cc marker
ny c th c xc nh trong mi mt c th bt k tui no thm
ch giai on bo thai nn chng rt c ch trong vic chn on sm cc
bnh di truyn. Tuy nhin cn lu rng mt marker c s dng
nh du trn mt NST no s c di truyn cng vi gene bnh ch
khng c ngha l n chnh l nguyn nhn ca bnh.
5. Lp bn gene da trn phn tch hin tng di truyn lin kt
Hn mt thp nin trc, vic phn tch lin kt rt t c c may
thnh cng v cc nh di truyn hc ch c trong tay vi chc marker a
hnh (polymorphic markers) hu dng cho vic tm kim hin tng lin
kt trong ton b genome ca ngi nh cc allele ca cc gene quy nh
cc nhm mu. Nh vy s khng chc rng mt gene bnh s nm gn
vi mt marker c th t ti c mt s lin kt c ngha hay
khng. Tnh trng ny thay i mt cch ngon mc trong hn mt
thp nin gn y, khi hng ngn marker a hnh mi (RFLP, VNTR v
STRP) c khm ph.

Hnh 2: Bn di truyn ca NST s 9 cho thy v tr ca mt lng ln cc


marker a hnh. V tn s ti t hp n thng cao hn nam nn khong cch
gia cc marker (tnh bng cM) ngi n ln hn so vi ngi nam.

Nh k thut xc lp kiu gene hiu qu v c mt lng ln cc


marker, vic lp bn cho mt gene bnh tr thnh mt cng vic bnh
thng v ch tiu tn mt thi gian ngn chng vi tun hoc vi thng
thng qua cc phn tch thng k v phn tch trong phng th nghim.
c th phc v cho vic lp bn gene cc marker phi c cc c

tnh sau:
(1) Chng phi ng tri (codominant), tc l c th d dng phn
bit c gia d hp t v ng hp t. iu ny gip d xc nh phase
lin kt (linkage phase). Cc RFLP, VNTR v STRP hon ton tho mn
tiu chun ny, trong khi cc loi marker c in nh nhm mu ABO,
nhm mu Rh khng c.
(2) Cc locus marker phi nhiu c th c c nhng marker
nm gn gene bnh. Hin nay chng ta c y cc locus nh vy
cho tt c cc NST. (hnh 2)
(3) Cc locus marker cng c ch khi chng c a hnh cng cao
tc l c nhiu allele khc nhau trong qun th. Mt mc a hnh cao
s m bo tt c cc b m s d hp t cc locus marker, iu ny to
u kin thun li hn trong vic xc lp phase lin kt trong cc gia
nh. S a hnh trong cc on lp vi v tinh to ra rt nhiu allele v rt
d nh gi, tnh cht ny lm chng tr thnh mt cng c ht sc hu
ch trong vic lp bn gene.
V d di y minh ha cho ng dng ca cc c im ny trong
vic lp bn gen:

Hnh 3: Ph h minh ha s di truyn ca mt bnh di truyn tri NST thng.


A, Mt marker RFLP gm 2 allele lin kt gn vi locus mang gene bnh c
lp cho tng thnh vin ca gia nh, nhng khng xc nh c phase lin kt.
B, Mt marker a hnh vi v tinh vi 6 allele lin kt gn vi locus mang gene
bnh c lp cho tng thnh vin ca gia nh, cho php xc nh d dng
phase lin kt

Trn ph h A hnh 3, mt ngi n ng mc bnh ng hp t


v 2 allele RFLP lin kt gn vi locus ca gene bnh (cn nh rng hu

ht cc RFLP xut hin l do s c mt hoc vng mt ca cc v tr gii


hn v do ch c 2 allele trong qun th). V ng ta d hp t. Con gi
mc bnh ca h ng hp t v allele marker. Da trn cc kiu gene,
ngi ta khng th xc nh phase lin kt trong th h ny, v th s
khng th d on c a tr no s b mc bnh v a tr no s
khng mc bnh . Cuc hn nhn th h th nht c gi l mt hn
nhn khng c thng tin (uninformative mate).
Tri li, mt a hnh ca on lp vi v tinh vi 6 allele c
phn loi trong cng mt gia nh (ph h B) s cho php xc nh d
dng phase lin kt. V ngi m trong th h I c 2 allele khc vi 2
allele ca ngi b mc bnh, do chng ta c th xc nh rng ngi
con gi mc bnh ca h th h II c truyn gene bnh qua mt
NST mang allele 1 ca marker. V c ta ly mt ngi chng c allele 4
v 5, chng ta c th d bo rng a con no ca h nhn allele 1 t c ta
s b mc bnh, trong khi a con no nhn alllele 2 s bnh thng, tr
trng hp xy ra ti t hp gene do trao i cho. V d ny cho thy gi
tr ca cc marker c tnh a hnh cao khng nhng trong vic phn tch
hin tng lin kt m cn trong vic chn on bnh di truyn.

II. Lp bn vt l (physical mapping)


Vic phn tch lin kt ch cho chng ta xc nh khong cch tng
i gia cc locus, nhng khng cho php xc nh cc v tr c hiu ca
cc marker hay cc gene bnh. Vic lp bn vt l cho php gii quyt
hn ch ny. Ti nay c nhiu tin b ng k v mt k thut cho php
thc hin c vic lp bn vt l vi phn gii cao.
1. Lp bn da trn hnh thi nhim sc th
Mt phng php trc tip v n gin lp bn ca cc gene
bnh l xc nh s kt hp hng nh gia mt bnh vi mt bt thng
di truyn t bo hc, nh nhn on, mt on hoc nhng bt thng
khc ca NST. Nhng bt thng nh vy c th t n khng c hu qu
trn lm sng (do n c th ng vai tr nh mt marker) hoc c th
gy bnh.
1.1. Tnh d hnh (heteromorphisms)
Tnh d hnh l mt bin d trong hnh thi ca mt NST. S d hnh
cng tng t nh tnh a hnh, chng l nhng bin d t nhin xy ra
gia cc c th trong qun th. Tuy nhin s khc bit c bn y l tnh
d hnh c th pht hin c bng knh hin vi quang hc cn tnh a
hnh khng th pht hin c bng phng tin ny.

Tnh d hnh cng tng t nh cc


locus marker, khng gy ra mt bin d hay
mt bnh di truyn no c nhng n c th
lun lun i km vi mt bin d hay mt
bnh di truyn no trong gia nh v qua
gip xc nh v tr ca gene. Tuy nhin
do khng ph bin nn trong vic lp bn
di truyn tnh d hnh ch c ch trong mt vi
trng hp.
1.2. Hin tng mt on (deletion)
Khc vi tnh a hnh, hin tng mt
on NST l nguyn nhn trc tip ca mt
s bnh di truyn. Karyotype ca nhng bnh
nhn mc bnh di truyn i khi cho thy c
lin quan n mt on ca mt s vng c
hiu trn mt NST. t bin ny cung cp
mt mi lin h mt thit gia v tr ca locus
mang gene bnh vi vng b mt on.
Mc mt on c th thay i gia
cc bnh nhn mc cng mt bnh. S mt
on c so snh gia cc bnh nhn khc
nhau c th xc nh vng b mt chung
gia tt c cc bnh nhn, v vy s thu hp
c vng c kh nng mang gene bnh
(hnh 4).
Vng mt on c s dng Hnh 4: nh v mt gene
lp bn da trn s mt on ca cc bnh thng qua hin tng
gene chu trch nhim cho bnh u nguyn mt on.
bo vng mc (retinoblastoma), hi chng Mt lot cc trng hp
Prader-Willi v Angelman, U Wilm (mt mt on gy ra cng mt
loi u ca thn tr em c th gy ra do t kiu bnh gi ln nhau
c nghin cu. V tr
bin trn NST s 11).
tng i ca gene bnh s
Cn lu l hin tng mt on ch nm trn vng gi ln nhau
nh hng trn mt trong hai NST tng ca 6 trng hp mt on
ng v bnh nhn c biu hin d hp t v
tnh trng mt on. Thng nu c mt vng b mt c kch thc ln
quan st bng knh hin vi quang hc xy ra trn c hai NST tng
ng s c tc dng gy cht cho cc c th mang kiu t bin .

1.3. Hin tng chuyn on (translocation)


Hin tng chuyn on cn bng thng khng gy hu qu trn
kiu hnh do vn gi c nguyn vn vt liu di truyn. Tuy nhin nu
t bin ny lm t ngang mt gene no n c th gy ra mt bnh di
truyn. Da vo c im ny c th xc nh c v tr tng i ca
gene bnh trn vng b t gy ca NST.
Bng vic phn tch lin kt lp c bn ca gene NF1 mt
cch phng chng trn nhnh di ca NST 17. V tr chnh xc hn ca
gene ny c xc nh da trn hai bnh nhn trong mt bnh nhn
mang chuyn on cn bng gia NST 17 v 22 v mt bnh nhn khc
mang chuyn on cn bng gia NST 17 v 1. Cc im gy ca nhng
chuyn on ny trn NST 17 nm rt gn vi vng c gi qua vic
phn tch lin kt. Hin tng ny cung cp mt im mc vt l cho
cc th nghim t dn n vic dng ha c gene ny.
2. Lp bn bng phng php lai ti ch (in situ hybridization)
Phng php lai ti ch (hnh 5) l mt phng php n gin cho
php lp bn ca cc on DNA mt cch trc tip. Do c phn gii
cao v d s dng nn k thut FISH c s dng rng ri lp bn
vt l ca gene.
Gi s chng ta c mt on DNA mang mt mt gene bnh m
chng ta mun bit v tr, DNA ny s c dng ha lm probe, sau
probe ny s c cho tip xc vi tiu bn mang cc NST k gia
vi cc DNA c tch xon. Probe gn hunh quang s bt cp theo
nguyn tc b sung vi DNA tch xon ca NST ti mt v tr c hiu
do c th nh v mt cch chnh xc v tr ca probe trn NST vi
phn gii khong 1Mb.
Bng cch s dng cc NST trong gian k, do trong k ny NST t
kt c hn so vi trong k gia nn phn gii trong k thut FISH s
c th ln ti 25 n 250kb.
K thut fiber FISH l mt ci tin ca k thut FISH, trong k thut
ny ngi ta s dng cc bin php ha hc v vt l tc ng ln cc
NST gian k ko dui si chromatin lm tng quai (loop)
chromatin c th nhn thy c. Vi k thut ny, cc probe c kch
thc ch mt vi kb cng c th c s dng.

Hnh 5: S dng phng php lai ti ch nh v mt on DNA trn


NST c hiu

3. K thut lai t bo sinh dng (somatic cell hybridazation)


K thut lai t bo sinh dng (hnh 6) l mt phng php nh v
gene trn NST da trn hin tng cc t bo sinh dng thuc cc loi
khc nhau nu c nui cy trong cng mt mi trng vi s c mt
ca cc tc nhn nh polyethylene glycol hay virus Sendai i khi s ha
nhp vi nhau to thnh cc t bo lai.
Khi nui cy t bo chut v ngi vi nhau theo cch ny, cc t
bo lai s cha 86 NST vi 46 NST ca ngi v 40 NST ca chut.
Nhng t bo ny s c a vo mi trng chn lc nh HAT
(hypoxanthine, amino-pterine, v thymidine) loi b nhng t bo
khng ha nhp. Cc t bo lai sau s c cho nhn ln, qua qu trnh
nguyn phn nhng t bo ny s bt u mt dn cc NST ngi trong
nhn t bo v hnh thnh nhng t bo c b NST y ca chut
nhng ch c mt hoc vi NST ca ngi.

Hnh 6: Lp bn
gene bng k thut lai
t bo sinh dng.
Cc t bo ca ngi
v t bo ca loi gm
nhm c ha ln v
chn lc. Cc t bo lai
c xu hng mt dn
cc t bo ngi qua
qu trnh nguyn phn,
kt qu l mi dng
(clone) ch mang 1
hoc vi NST ngi.
Mi dng c nh
gi xt xem c 1 loi
gene nht nh no
hay khng, qua xc
nh c gene nm
trn NST no

Cc t bo khi s c lp karyotype xc nh ang mang


NST no ca ngi (cc NST ca ngi v chut c phn bit vi nhau
thng qua kch thc v cc mu band trn NST). Nhng nhm t bo ny
sau c nghin cu xc nh xem nhm no lun lun mang gene
no (Hnh 7).

Hnh 7: Bng phn tch lai t bo sinh dng. Lu l on DNA ang c


nh gi cho thy c s kt hp vi clone 1, 3, 4, v 7. Tt c cc dng ny u
cha NST s 9 trong khi cc dng 2, 5, 6 v 8 khng mang NST ny. Kt qu
phn tch ny cho php kt lun on DNA ny nm trn NST s 9.

V d nu gene lun lun c thy nhm t bo mang NST s 1


ca ngi nhng khng bao gi thy cc nhm t bo mang cc NST

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khc th chng ta c th kt lun rng gene phi nm trn NST


s 1.
S c mt ca gene trong t bo lai c th c xc nh theo nhiu
cch khc nhau:
(1) Nu ngi ta bit mt enzyme (do mt gene no cha bit
v tr m ha) th vic phn tch ezyme s c tin hnh trn cc dng
t bo. in di protein c th c s dng pht hin mt sn phm
protein ca ngi v phn bit n vi protein tng ng ca chut. Hng
nghin cu ny i hi phi pht hin c protein bit trong cc
dng t bo.
(2) Hin nay ngi ta thng dng ph bin phng php lai gia
DNA ca mt dng t bo lai vi mt probe c nh du mang on
DNA quan tm v sau s dng k thut Southern blotting hoc k thut
PCR pht hin NST no mang on DNA (hnh 8).

Hnh 8: K thut Southern Blotting c s dng trong vic lp bn gene


bng k thut lai t bo sinh dng (i chiu vi hnh 8). S khc nhau trong v
tr ct gii hn dn n s khc nhau trong kch thc ca cc on DNA gia
ngi v chut. Probe ca gene ngi ch lai vi cc dng t bo lai 1, 3, 4 v 7
chng t probe ch lai khi c s c mt ca NST s 9.

4. Lp bn bng phng php lai phng x (radiation hybrid mapping)


y l mt k thut hiu qu v c s dng mt cch rng ri
(hnh 9). Phng php ny c bt u bng mt t bo lai sinh dng
ch mang mt NST ca ngi. Cc t bo ny c chiu x bng tia X
hoc tia gamma gy ra nhiu ch t trn chui xon kp trn cc NST.
Nhng on NST ngi b t ra ny c th tip tc tn ti chng phi

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ho nhp vi cc NST
ca loi gm nhm.
Cc t bo sau khi
ho nhp c gi l
cc t bo lai do chiu
x (radiation hybrids)
mang cc on nh
NST ngi ho nhp
vi NST ca loi gm
nhm. S c mt ca
cc NST ngi trong
nhng t bo ny c th
c pht hin bng
cch sng lc cc on
Alu (c vi kb trn
NST ngi c th gp
cc on lp Alu nhng
li khng thy trn NST
ca loi gm nhm).
V vic chiu x
gy t gy trn NST
ngi nhng khong
Hnh 9: K thut lp bn gene bng phng
php lai phng x. Mt dng t bo lai ch cha cch ngu nhin do
mt NST ngi c chiu x gy t gy trn cc locus cng nm gn
NST , nhng on t gy ny s ha nhp vi nhau bao nhiu s c
NST ca loi gm nhm tn ti. Cc locus gn gp nhiu hn by
nhau nh A v B s thng c thy trn cng nhiu so vi cc locus
mt on NST, trong khi nhng locus xa nhau nm xa nhau trn cng
nh A v C s t khi c thy nm trn cng mt mt on NST.
NST.
Cc k thut nh
PCR c th c dng xc nh nhng t bo lai do phng x no mang
cc kt hp c hiu ca cc locus ca ngi (ngha l cc on mi PCR
c hiu cho locus s c s dng khuch i locus nu n c mt
trong t bo). Sau cc k thut thng k c s dng nh gi tng
cp locus thng c gp trn cng mt on NST qua cung cp
mt nh gi v khong cch tng i gia cc locus. Ngi ta cng gia
tng mc chiu x to ra nhng on nh hn v nh c th tng
phn gii ca k thut.

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5. Dng ha theo v tr (positional cloning)


Trong nghin cu di truyn i khi sn phm ca mt gene chu
trch nhim cho mt bnh di truyn no c bit trc khi gene c
xc nh.
V d trong trng hp bnh hng cu hnh lim, ngi ta bit
nguyn nhn ca bnh l do bt thng trn chui polypeptide beta globin. T trnh t ca cc amino acid trn chui polypeptide ny ngi ta
c th suy ra trnh t ca DNA, trnh t ny c s dng tng hp
probe nhn to. Cc probe ny s c dng nh v gene bnh theo cc
k thut nh c m t trn.
Cch xc nh v tr ca gene da trn sn phm ca gen v chc
nng ca sn phm ny c gi l k thut dng ha da vo chc nng
(functional cloning).
Tuy nhin nh v cc gene thng chng ta phi da vo phn
tch hin tng lin kt gene, vi phng php ny gene bnh ch c
nh v trong vng c kch thc khong 1Mb hoc ln hn. Vi kch
thc ny nhng vng c th cha ti hng chc gene, xen k vi
nhng vng DNA khng mang m. c th xc nh chnh xc v tr ca
mt gene bnh trn vng ngi ta phi thc hin vic phn tch t mt
marker lin kt (linked marker) trn vng ri sau phn tch ln ti cc
vng hai bn marker ny nh v v dng ha gene bnh. K thut ny
c gi l k thut dng ha theo v tr (positional cloning).
Gi s rng chng ta bit v tr ng chng ca mt marker a hnh
thi lin kt cht ch vi mt gene bnh m chng ta mun phn lp
(ngha l chng nm cch nhau khong 1Mb). lm c vic ny
ngi ta dng mt k thut c gi l "i dc NST" (chromosome
walking) tin dn v pha gene bnh. on DNA ca marker c
dng lm probe nht ra cc on gi u ln nhau mt phn trong th
vin genome. Cc on gi u c th c xc nh bng k thut PCR,
qua k thut ny s cho thy mt phn ca probe v ca on DNA tch t
th vin genome u c th khuch i bng cng cc on mi (primer)
ging nhau (hnh 11).
Ngay sau khi c xc nh, on gi u DNA va c ly ra t
th vin genome c s dng tr li nh mt probe nht tip mt
on DNA khc gi u mt phn vi n pha u kia t th vin
genome. Bng cch ny ngi ta ln lt dng cc on DNA gi mt
phn ln nhau i dc trn NST ht on ny ti on khc cho ti khi
i n c gene bnh. Do i khi chng ta khng chc gene bnh nm
pha bn ny hay pha bn kia ca marker nn k thut i dc NST s

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c tin hnh c hai pha.

Hnh 10: Mt probe c kim tra qua 8 dng t bo ly t th vin YAC. Probe
lai vi hai trong s 8 dng (dy 6 v 7), chng t c s gi u gia DNA
ca ca probe v DNA mi mt trong hai dng YAC thuc clone 6 v 7.

Vic xc nh on gi u gia cc on DNA hin nay c thc


hin kh d dng thng qua vic nh v hng chc ngn v tr c gi l
cc v tr ni on (sequence tagged sites: STSs) v tng hp c chng
bng con ng nhn to, STSs l cc on di vi trm bp nm ti cc v
tr c xc nh trn NST. Chng nm cnh cc primer PCR bit,
do bt c phng xt nghim no cng c th dng k thut PCR
khuch i chng ln (nh cc primer c hiu) phc v cho vic phn
tch. Bng cch ny chng tr thnh cc "bin bo" cho nhng on DNA
bit trn ton b genome m s gi u nhau ca cc STS gia cc
on DNA khc nhau trong th vin genome s cho php lp ghp chng
li vi nhau theo mt trnh t nht nh (hnh 11). Cc STS c nh v
trn cc NST c hiu bng cch s dng cc k thut nh lp bn
bng phng php lai phng x.
Cc on DNA c dng trong k thut "i dc NST" c th c
dng ha thnh cc vector nh plasmid, cosmid hoc NST nhn to ca
nm men (yeast artificial chrromosome: YACs). Cc plasmid thch hp
i vi vic ci cc on DNA nh nht, trong khi YACs thch hp cho
cc DNA ln nht vi kch thc t 200 - 2000kb.

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Cc vector khc
uc dng ha c th n
nh hn nhng mang
on ci DNA nh hn
nh: cc NST nhn to
ca vi khun (bacterial
artificial
chromosome:
BACs); NST nhn to ca
th thc khun P1
(bacterialphage
P1
artificial chromosomes :
PACs), c hai vector ny
u tip nhn c cc
on DNA di vi kch
thc t 50 - 200 kb.
Chng thng c dng
kt hp vi YACs trong
k thut dng ha theo v
tr.
Hnh 12 m t mt bn
ca cc on YAC v
cosmid gi u nhau trong
vng mang gene gy bnh
polyp i trng u tuyn
(adenomatous polyposis

Hnh 11: S dng cc STS xc nh cc v tr


gi u gia cc on DNA trong k thut "i
dc NST". S gi u c xc nh khi cc
primer ca mt STS c hiu oc s dng
khuch i DNA t cc on DNA khc nhau
ly ra t th vin genome

Hnh 12: Mt v d v "contig map" ca cc YAC c s dng trong k


thut "i dc". NST s 5 trong vng mang gene gy bnh polyp i trng u
tuyn

15

coli). Bn ca cc on DNA k tip (contiguous DNA) nhau nh th


ny c gi l "contig map".
Tm li k thut "i dc NST" lin quan n vic phn lp cc on
DNA gi u nhau mt phn (partial overlapping DNA segments) t th
vin genome i dc trn NST v pha v tr ca gene bnh.
Khi vic "i dc NST" hon tt, lm th no m chng ta bit l
mnh ti c gene bnh ? bit c iu ny ngi ta p dng cc
cch sau:
5.1. Phn tch s bo tn on DNA mang m gia cc loi (Cross Species Conservation)
Khi i dc trn mt on DNA tm kim mt gene no , chng
ta mun trnh nhng phn khng mang m tuy nhin phn ln on DNA
ny li l phn khng mang m. Phn DNA mang m m chng ta mun
tm nhn chung khng thay i nhiu trong lch s tin ha do chc nng
quan trng ca n i vi s sng cn ca sinh vt, do vy n c
bo tn v gii thch v sao trnh t DNA li c s tng ng gia mt s
ln cc loi khc nhau. Tri li nhng on khng mang m thay i
nhanh chng v c s khc bit rt ln gia cc loi. Nh vy nu trnh t
m chng ta ang xem xt trong k thut "i dc NST" gm nhng on
mang m, chng c th lai vi cc on DNA ca cc loi khc (theo kiu
b sung gia cc base) v ngc li.
nh gi xem mt on DNA c c bo tn trong cc loi
khng, mt probe mang mt on DNA c tng hp v cho tip xc
vi cc on DNA tho xon thuc cc loi khc nhau xem c xy
ra qu trnh lai hay khng, nu c th on DNA ny c kh nng l mt
on DNA mang m (phng php ny c gi la "zoo blot"). Mc d
iu ny t n khng gip xc nh v tr gene bnh nhng n gip d bo
kh nng on DNA m chng ta ang kho st l mt vng mang m ca
gene.
5.2. Xc nh cc o CG (Identification CG Islands)
Nh c gii thiu trong cc bi trc, hu ht cc dinucleotide
CG u c methyl ha. Tuy nhin khong 60% gene ngui, bao gm
gn nh tt c cc gene qun gia (housekeeping genes) v hu ht cc
gene hot ng, u c cc dinucleotide CG khng b methyl ha
(unmethylated) u 5'. Nhng dinucleotide ny c gi l cc o CG
(CG island). C l khi khng b methyle ha u 5' s lm gene d tip
xc hn vi cc yu t thc y qu trnh sao m.

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Khi chng ta xc nh c mt dy cc o CG c ngha l nh


v c mt gene m ha.
6. Cc k thut xc nh on exon
Nhiu k thut hin
nay ang c dng xc
nh cc on exon trong
trnh t DNA ca genome.
Mt trong nhng k thut
ny c gi l k thut
by exon (exon trapping).
on DNA ca ngui c
t vo trong mt vector
plasmid bng k thut
DNA ti kt hp. Vector
plasmid c dng ha
trong t bo ca loi c v
hay ca nm men c mang
h thng sao m thch hp.
Phn t mRNA hon chnh
c phn lp v chuyn
thnh cDNA. cDNA sau
c khuch i bng k
thut PCR xc nh
chiu di. Nu on DNA
ca ngi c cha mt
hoc nhiu on exon,
cDNA s c kch thc di
hn so vi trng hp
khng c.
Hnh 13: K thut by exon.
Trong k thut ny
mt exon hay nhiu exon
trong mt on DNA c pht hin bng cch ci on DNA vo
trong mt vector plasmid bng k thut DNA ti t hp m nhng vector
ti t hp ny cho php on DNA sao m trong cc t bo ca loi c
v hoc nm men (hnh 13). Khi qu trnh hon thin mRNA xy ra tt c
cc on RNA khng mang on exon s b ct. Nu on DNA m chng
ta ang kho st c cc mang cc on exon th sau khi hon thin mRNA
s c kch thc di hn (do c mang cc exon ca ngi t on DNA
ci vo), nhng nu on DNA m chng ta kho st khng c cc on

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exon th mRNA sau khi hon thin s c kch thc ngn hn.
Mt k thut pht hin exon ph bin khc c gi l k thut chn
lc cDNA trc tip (direct cDNA selection). Trong k thut ny cc on
DNA ca genome c a vo trong YAC. Cc YAC sau c lai vi
cc dng cDNA c ly t th vin cDNA ca ngi. Nu on DNA
trong YAC c mang cc exon, chng s bt cp theo nguyn tc b sung
vi cc on trong th vin cDNA. Do cDNA c lm t cc on DNA
m ha thng qua cc mRNA hon chnh nn nu on DNA khng
mang cc exon, chng s khng bt cp c vi cDNA. Sau mt vi chu
k lai cc dng cDNA tng ng vi cc exon trong on DNA s c
lm giu cho php xc nh chng. Mt nhc im ca phng
php ny l n c th khng pht hin c cc gene him v khng c
trong th vin cDNA.
7. Phn tch trnh t DNA bng k thut vi tnh
Vic phn tch bng k thut vi tnh (cn c gi l nghin cu in
silico) em li mt phng php hu hiu cho vic xc nh gene. Cc
phn mm vi tnh phc tp nh GRAII, c th kim tra mt on DNA
xc nh s c mt ca nhng v tr trng yu nh v tr bt u sao m,
cc codon kt thc, cc on ranh gii gia intron - exon v.v... t
xc nh vai tr mang m ca gene.
Bin php ny c dng xc nh v m t c im ca
mt trong s cc gene gy bnh thn a nang ngui trng thnh (adult
polycystic kidney disease gene).
Hn na, cc phn mm ny cn ghi nhn cc mu gene in hnh
m cho cc loi protein c hiu nh cc yu t sao m, cc protein mng
t bo v.v...
C s d liu (database) v nhng on DNA bit c vi tnh
ha ng mt vai tr quan trng trong vic xc nh gene. Khi nghin cu
mt on no ca DNA tm mt gene, ngi ta s tm s tng ng
gia on DNA ca vng ny vi cc thng tin c lu tr v trnh t
ca DNA bit trong c s d liu ca my. Cc thng tin ny c ly
t cc gene bit chc nng hoc t cc mRNA c tng hp ti cc
m c hiu.
Gi s chng ta s dng vic phn tch lin kt xc nh mt
vng cha mt gene gy ra s ri lon pht trin nh d dng ca chi
chng hn. Khi thc hin k thut "i dc NST" chng ta s tm kim s
tng ng gia 1 on DNA ly t vng ny vi mt on DNA c trong
c s d liu nh trnh t DNA ca mt gene m cho mt protein lin

18

quan n s pht trin ca xng nh mt yu t pht trin nguyn bo


si (fibroblast growth factor). V cc gene m ha cho cc sn phm
protein tng t thng c trnh t DNA tng t. S tng ng trong
trnh t DNA gia on DNA ly t vng ny vi mt on trong c s
d liu gi on DNA ny thc s l mt phn ca gene gy d dng chi.
Cc c s d liu c dng trong vic tm kim cc on tng
ng thng l trnh t ca cc on DNA nh c gi l expressed
sequence tags (ESTs). ESTs thu c t vic c trnh t ca hng trm
bp t hai u ca cc dng cDNA ly t th vin cDNA. V cc clone ny
ch gm cc DNA b sung chnh xc vi mRNA nn ESTs i din cho
cc phn biu hin (expressed) ca gene. Thng chng ch biu hin
mt s m nht nh nhng thi im nht nh.
Cc EST c th c lp bn trn cc NST c hiu bng cch s
dng cc k thut lp bn vt l c m t phn trc nh lp
bn gene bng k thut lai phng x (radiation hybrid mapping) hoc
k thut FISH. Khi cc nh nghin cu c th tm kim trnh t tng
ng gia cc on DNA ti mt vng ang c quan tm vi cc ESTs
bit. Nu c s tng ng, v tr ca on DNA trn NST s cng
mt vng vi ESTs v qua cng c th bit loi m lin quan ti
loi bnh ang kho st (ni gene hot ng sao gii m). Vic xc
nh cc ESTs v ang c thc hin mt cch nhanh chng v hin
c gn 2 triu ESTs trong cc d liu trn my. K thut ny c
s dng xc nh mt trong s cc gene gy bnh Alzheimer.
Vic tm kim s tng ng v trnh t khng nht thit i hi
phi s dng gene ngi. Ngi ta thy c s tng ng gia trnh t ca
mt s gene bit chc nng ngi vi cc gene ca chut nht hoc
thm ch ca nm men hoc vi khun . V cc gene m ha cho cc sn
phm protein quan trng c xu hng c bo tn trong qu trnh tin
ha nn s xc nh s tng ng gia gene ngi vi gene ca mt sinh
vt khc c th cung cp nhng thng tin quan trng v chc nng ca
gene ngi. V d nhiu gene lin quan n vic u ha chu k sinh
sn t bo hon ton tng t gia ngi v nm men nh gia cc on
ca gene NF1 ngi vi gene IRA2 ca nm men.
C khong mt phn ba s gene gy bnh ngi c xc nh
l c s tng ng vi cc gene nm men. Cc gene ny v cc sn
phm ca n c th c th nghim mt cch d dng trn cc sinh vt th
nghim. Qua nhng hiu bit v chc nng ca chng trn cc sinh vt th
nghim s gip hiu bin tt hn v chc nng ca chng trn ngi. Mt
s cc gene bnh quan trng ngi c xc inh bi v c cc gene

19

gy ra biu hin tng ng c xc nh trc cc sinh vt khc.


V d s tng ng gia gene gy bnh mt nh chut v gn gy tt
khng c mng mt (aniridia) ngi.
8. Sng lc cc t bin trn trnh t DNA
Ngay khi mt phn ca DNA mang m c phn lp, n c th
c nh gi cc t bin bng cch s dng cc k thut nh SSCP
(single - strand conformation polymorphism), DGGE (denature gradient
gel electrophoresis) v c trnh t DNA trc tip (direct DNA sequence)
v.v.... Nu mt on DNA i din cho gene bnh th n ch thy cc c
th mang bnh ch khng th thy nhng ngi bnh thng.
gip phn bit cc t bin gy bnh vi cc tnh a hnh vn c
mt mt cch bnh thng mi c th, ngi ta so snh DNA ca bnh
nhn mc bnh do mt t bin mi gy ra vi DNA ca b m khng
mc bnh. Trong khi tnh a hnh khng c hi s c thy c b m
khng mc bnh v c con ci mc bnh, mt t bin mi gy bnh s ch
thy con m khng thy b m. y l phng cch hu ch trong vic
xc nh cc t bin gy ra cc bnh di truyn tri NST thung c tnh
thm cao nh bnh u x thn kinh type 1 (NF1).
Mt loi t bin khc c th c nh gi l cc trng hp mt
on di hin vi (submicroscopic deletion), y l nhng mt on qu
nh do khng th quan st bng knh hin vi c. Cc mt on ny
ch c th c pht hin bng cch s dng k thut Southern Blot. Mt
mt on nh vy s lm hnh thnh mt on gii hn (restriction
fragment) nh hn bnh thng lm n di chuyn nhanh hn trn gel.
i khi cc mt on ln hn c th c pht hin thng qua k
thut in di trn gel ging nh trong k thut Southern Blot nhng ci
tin bng vic ct gii hn (restriction digest) c thc hin bng cc
enzyme ct v tr gii hn (recognition site) him gp do s ln DNA
b ct s t i, do vy s lm xut hin cc on gii hn c chiu di t
vi chc n vi trm kb. Nhng on ny do c kch thc qu ln nn
kh phn bit vi nhau khi thc hin in di trn gel theo cch thng
thung nhng bng cch thay i chiu ca xung in ngang qua gel
(xung sau c thc hin gc 900 so vi xung trc), cc on ny s di
chuyn mt cch khc nhau ph thuc vo kch thc ca chng v cho
php phn bit chng d dng hn. V vy phng php in di ny c
gi l phng php in di trng xung in ( pulsed field gel
electrophoresis).

20

9. Test pht hin s hot ng sao m ca gene


kim tra xem mt gene c phi n chu trch nhim cho mt
bnh no khng, ngi ta c th kim tra cc m khc nhau xc
nh xem gene hot ng sao m m no. Vic kim tra c thc
hin bng cch tinh khit mRNA chit t mt loi m no , t n trn
ming thm (blot) v cho lai vi mt probe lm t gene ny pht hin.
K thut ny c gi l Northern blotting ( mun ni tng t nh k
thut Southern blotting nhng thay v cc on DNA th y l mRNA)
(hnh 14). Nu gene ny chnh l nguyn nhn gy bnh th phn t
mRNA ca n s c mt cc m l chu nh hng ca bnh (nguyn l
ca phng php ny cng ging nh nguyn l ca vic phn tch bng
ESTs ).
V d enzyme phenylalanine hydroxylase c tng hp gan do
gene m cho phenylalanine hydroxylase (t bin s gy bnh phenylketonuria, PKU) s hot ng sao m t chc ny.

Hnh 14: Mt v d v k thut Northern Blot. Trn nh cho thy hin tng lai
gia mt probe cDNA t gene EVI2A (mt gene ci vo trong mt on intron
ca gene NF1) vi mRNA c ly t tuyn thng thn, no, v cc nguyn
bo si. Kt qu cho thy EVI2A c biu hin no mc cao hn hai
loi m kia.

Mt test khc cho php nh gi trc tip s biu hin ca gene l


a mt on DNA bnh thng vo trong mt t bo b khim khuyt ly
ra t mt c th mc bnh (hoc t cc m hnh bnh tng t sinh vt

21

th nghim) bng k thut DNA ti kt hp. Nu on DNA bnh thng


ny khc phc c khim khuyt ca t bo c ngha l n c th i
din cho gene bnh m chng ta quan tm. K thut ny c dng
chng minh cc t bin trn gene CFTR c th gy bnh x nang (cystic
fibrois).
Tuy nhin vic dng ha cc gene i khi rt phc tp v kh khn
do nhiu yu t khc nhau, chng hn nh vic phn lp cc on DNA
gi u (overlapping) c th tr nn rt kh khn khi gp phi cc on
lp di trn DNA . Nhng kh khn ny lm cho vic dng ha cc gene
tr thnh mt cng vic tiu tn nhiu thi gian v cng sc.
Mc du gene gy bnh Huntington c xc nh l nm trn
nhnh ngn ca NST s 4 t nm 1983 nhng phi mt 10 nm lm vic
vt v ca mt nhm gm nhiu nh nghin cu gene ny mi c dng
ha. Vic dng ha gene gy bnh x nang (cystic fibrosis) cng mt 4
nm.
Vic dng ha cc gene hin c tin hnh vi nhiu thun li
hn nh cc k thut dng ha tt hn v cc vector mi c pht minh
(nh BACs, PACs v YACs c kh nng ci cc on DNA ln hn nhiu
so vi cc cosmid). Vic s dng cc mc vt l dng lp bn gene
(STSs) cng lm tng tc v hiu qu ca vic dng ha gene.
10. Cc gene ng vin (candidate genes)
Vic sn tm gene c tin hnh hiu qu hn nhiu khi c sn
mt gene ng vin. Nh tn gi y l mt gene m sn phm protein ca
n lm n tr thnh mt ng vin cho mt bnh ang cn tm nguyn
nhn. V d nh cc gene m cho cc loi collagen khc nhau c xem l
cc ng vin c th l nguyn nhn ca hi chng Marfan v collagen l
mt thnh phn quan trng ca t chc lin kt. Tuy nhin vic phn tch
lin kt bng cch s dng cc marker ca gene collagen trn cc gia nh
mc hi chng ny khng em li kt qu.
Khi gene fibrilline - 1 c xc nh trn NST 15, n tr thnh
mt gene ng vin cho hi chng ny (fibrilline cng l mt thnh phn
ca m lin kt). Vic phn tch lin kt cng nh v c gene gy
hi chng Marfan trn NST 15 v vy gene fibrilline - 1 tr thnh mt ng
vin c nhiu kh nng gy ra hi chng .
Vic phn tch cc t bin trn gene fibrilline - 1 chng t
nhng t bin ny kt hp mt cch hng nh vi hi chng Marfan v
cho php khng nh nhng t bin ny l nguyn nhn gy bnh.

22