LDLR oligonucleotides
The oligonucleotides found in the Hobbs et al, 1992 and Leitersdorf et al, 1990 paper are found on the gene map but many oligonucleotides have been published since. Some are listed in tables below:
Table 1: Hobbs et al, 1992; Hum Mutat 1:445. Marked on gene map.
Table 2: Leitersdorf et al, 1990; J Clin Invest 85:1014. Marked on gene map.
Table 3: Combination of Hobbs, Leitersdorf & Gudnason oligonucleotides which amplify at one PCR condition (Day et al, 1997; Hum Mutat 10:116). Marked on gene map.
Table 4: Lombardi et al, 1995; J Lipid Res 36:860. Oligonucleotide sequences for the amplification of LDLR using DGGE.
Table 5: Nissen et al, 1995; Hum Mutat 8:168. Oligonucleotide sequences for the amplification of LDLR using DGGE.
Table 6: Ekstrom et al, 1998, E J Clin Investig 28:740. Oligonucleotide sequences for the amplification of LDLR using DGGE.
Table 7: Heath et al, unpublished.
Table 1: Oligonucleotide sequences for the amplification of LDLR (Hobbs et al, 1992; Hum Mutat 1:445). Marked on gene map.
|
Region/ exon |
Oligonucleotide sequence (Sense & antisense, 5’ to 3’) |
Size (bp) |
|
Promoter |
FH1 GAG TGG GAA TCA GAG CTT CAC GGG T FH20 CCA CGT CAT TTA CAG CAT TTC AAT G |
155 |
|
Exon 1 |
FH2 ACT CCT CCC CCT GCT AGA AAC CTC A FH21 TTC TGG CGC CTG GAG CAA GCC TTA C |
234 |
|
Exon 2 |
FH3 CCT TTC TCC TTT TCC TCT CTC TCA G FH22 AAA ATA AAT GCA TAT CAT GCC CAA A |
172 |
|
Exon 3 |
FH4 TGA CAG TTC AAT CCT GTC TCT TCT G FH23 ATA GCA AAG GCA GGG CCA CAC TTA C |
176 |
|
Exon 4 (4A - 5’end) |
4AF GTT GGG AGA CTT CAC ACG GTG ATG G 4AR ACT TAG GCA GTG GAA CTC GAA GGC C |
355 |
|
Exon 4 (4B - 3’end) |
4BF CCC CAG CTG TGG GCC TGC GAC AAC G 4BR GGG GGA GCC CAG GGA CAG GTG ATA G |
267 |
|
Exon 5 |
FH5 CAA CAC ACT CTG TCC TGT TTT CCA G FH24 GGA AAA CCA GAT GGC CAG CGC TCA C |
173 |
|
Exon 6 |
FH6 TCC TTC CTC TCT CTG GCT CTC ACA G FH25 GCA AGC CGC CTG CAC CGA GAC TCA C |
174 |
|
Exon 7 |
FH7 AGT CTG CAT CCC TGG CCC TGC GCA G FH26 AGG GCT CAG TCC ACC GGG GAA TCA C |
169 |
|
Exon 8 |
FH8 CCA AGC CTC TTT CTC TCT CTT CCA G FH27 CCA CCC GCC GCC TTC CCG TGC TCA C |
175 |
|
Exon 9 |
FH9 TCC ATC GAC GGG TCC CCT CTG ACC C FH28 AGC CCT CAT CTC ACC TGC GGG CCA A |
271 |
|
Exon 10 (5’end) |
FH10 AGA TGA GGG CTC CTG GTG CGA TGC C FH29 GCC CTT GGT ATC CGC AAC AGA GAC A |
202 |
|
Exon 10 (3’end) |
FH11 GAT CCA CAG CAA CAT CTA CTG GAC C FH30 AGC CCT CAG CGT CGT GGA TAC GCA C |
162 |
|
Exon 11 |
FH12 CAG CTA TTC TCT GTC CTC CCA CCA G FH31 TGG GAC GGC TGT CCT GCG AAC ATA C |
168 |
|
Exon 12 |
FH13 GCA CGT GAC CTC TCC TTA TCC ACT T FH32 CAC CTA AGT GCT TCG ATC TCG TAC G |
209 |
|
Exon 13 |
FH14 GTC ATC TTC CTT GCT GCC TGT TTA G FH33 GTT TCC ACA AGG AGG TTT CAA GGT T |
217 |
|
Exon 14 |
FH15 CCT GAC TCC GCT TCT TCT GCC CCA G FH34 CGC AGA AAC AAG GCG TGT GCC ACA C |
202 |
|
Exon 15 |
FH16 GAA GGG CCT GCA GGC ACG TGG CAC T FH35 GTG TGG TGG CGG GCC CAG TCT TTA C |
246 |
|
Exon 16 |
FH17 CCT CAC TCT TGC TTC TCT CCT GCA G FH36 CGC TGG GGG ACC GGC CCG CGC TTA C |
127 |
|
Exon 17 |
FH18 TGA CAG AGC GTG CCT CTC CCT ACA G FH37 GCT TTC TAG AGA GGG TCA CAC TCA C |
207 |
|
Exon 18 |
FH19 TCC GCT GTT TAC CAT TTG TTG GCA G FH37 AAT AAA ACA AGG CCG GCG AGG TCT C |
135 |
PCR conditions: 1.5mM MgCl2.
Cycle conditions: (96oC 1min, 68oC 5min) x30.
Table 2: Oligonucleotide sequences for the amplification of LDLR (Leitersdorf et al, 1990; J Clin Invest 85:1014). Marked on gene map.
|
Region/ exon |
Primer |
Oligonucleotide sequence (Sense & antisense, 5’ to 3’) |
Size (bp) |
Cycle conditions |
|
Prom |
FH110 FH130 |
CAG CTC TTC ACC GGA GAC CC ACC TGC TGT GTC CTA GCT GG |
277 |
94(1),57(1),74 (1) |
|
Exon 1 |
FH111 FH131 |
CAC ATT GAA ATG CTG TAA ATG ACG CTA TTC TGG CGC CTG GAG CAA GCC |
215 |
94(1),59(1),74 (2) |
|
Exon 2 |
FH112 FH132 |
TTG AGA GAC CCT TTC TCC TTT TCC GCA TAT CAT GCC CAA AGG GG |
183 |
94(1),57(1),74 (1) |
|
Exon 3 |
FH113 FH133 |
TTC CTT TGA GTG ACA GTT CAA TCC GAT AGG CTC AAT AGC AAA GGC AGG |
196 |
94(1),57(1),74 (1) |
|
Exon 4 (5’end) |
FH114 FH134 |
GTG GTC TCG GCC ATC CAT CC AGC CAT CTT CGC AGT CGG GG |
242 |
94(1),63 (1),74 (2) |
|
Exon 4 (3’end) |
FH115 FH135 |
CCC CCA GCT GTG GGC CTG CG CGC CCC CAC CCT GCC CCG CC |
237 |
94(1),66(1),74 (2) |
|
Exon 5 |
FH116 FH136 |
AGA AAA TCA ACA CAC TCT GTC CTG GGA AAA CCA GAT GGC CAG CG |
180 |
94(1),58(1),74 (1) |
|
Exon 6 |
FH117 FH137 |
TCC TCC TTC CTC TCT CTG GC TCT GCA AGC CGC CTG CAC CG |
179 |
94(1),56(1),74 (1) |
|
Exon 7 |
FH118 FH138 |
GGC GAA GGG ATG GGT AGG GG GTT GCC ATG TCA GGA AGC GC |
236 |
94(1),57(1),74 (1) |
|
Exon 8 |
FH119 FH139 |
CAT TGG GGA AGA GCC TCC CC GCC TGC AAG GGG TGA GGC CG |
220 |
94(1),64(1),74 (2) |
|
Exon 9 |
FH129 FH140 |
CCC CTG ACC TCG CTC CCC GG GCT GCA GGC AGG GGC GAC GC |
224 |
94(1),63(1),74 (2) |
|
Exon 10 |
FH121 FH141 |
ATG CCC TTC TCT CCT CCT GC AGC CCT CAG CGT CGT GGA TA |
278 |
94(1),58(1),74 (2) |
|
Exon 11 |
FH122 FH142 |
TCC TCC CCC GCC CTC CAG CC GCT GGG ACG GCT GTC CTG CG* |
194 |
94(1),65(1),74 (2) |
|
Exon 12 |
FH123 FH143 |
ACT GGC ATC AGC ACG TGA CC CGT GTG TCT ATC CGG CCA CC |
236 |
94(1),58(1),74 (2) |
|
Exon 13 |
FH124 FH144 |
GTC ATC TTC CTT GCT GCC TG TTC CAC AAG GAG GTT TCA AGG TTG GGG GGG |
329 |
94(1),59(1),74 (3) |
|
Exon 14 |
FH125 FH145 |
AAA TTT CTG GAA TCT TCT GG GCA GAG AGA GGC TCA GGA GG |
268 |
94(1),59(1),74 (5) |
|
Exon 15 |
FH126 FH146 |
AGA AGA CGT TTA TTT ATT CTT TC GTG TGG TGG CGG GCC CAG TCT TT |
221 |
94(1),55(0.5),74 (1.5) |
|
Exon 16 |
FH127 FH147 |
CCT TCC TTT AGA CCT GGG CC CAT AGC GGG AGG CTG TGA CC |
173 |
94(1),58(1),74 (2) |
|
Exon 17 |
FH128 FH148 |
GGG TCT CTG GTC TCG GGC GC GGC TCT GGC TTT CTA GAG AGG G |
242 |
94(1),58(1),74 (2) |
|
Exon 18 |
FH129 FH149 |
GCC TGT TTC CTG AGT GCT GG TCT CAG GAA GGG TTC TGG GC |
135 |
94(1),56(1),74 (2) |
All PCR reactions consisted of 35 cycles and were completed by an extension of 10 min at 74oC.
* The first base in the 5’end is not on the gene map.
Table 3: Oligonucleotide sequences for the amplification of LDLR exons at one PCR condition. Marked on gene map.
|
Region/ exon |
Oligonucleotide sequence (Sense & antisense, 5’ to 3’) |
Size (bp) |
|
Prom |
FH110 CAG CTC TTC ACC GGA GAC CC FH130 ACC TGC TGT GTC CTA GCT GG |
277 |
|
Exon 1 |
FH2 ACT CCT CCC CCT GCT AGA AAC CTC A FH131 CTA TTC TGG CGC CTG GAG CAA GCC |
238 |
|
Exon 2 |
FH112 TTG AGA GAC CCT TTC TCC TTT TCC FH132 GCA TAT CAT GCC CAA AGG GG |
183 |
|
Exon 3 |
FH113 TTC CTT TGA GTG ACA GTT CAA TCC FH133 GAT AGG CTC AAT AGC AAA GGC AGG |
196 |
|
Exon 4 (5’end) |
FH51 GGTCTCGGCCATCCATCCCTG FH52 CTGTTGCACTGGAAGCTGGCGG |
242 |
|
Exon 4 (mid) |
FH53 AGACGAGGCCTCCTGCCCGGT FH54 GAGCAGGGGCTACTGTCC |
180 |
|
Exon 4 (3’end) |
FH55 CGACTGCGAAGATGGCTCG FH169 GGAACCCAGGGACAGGTGATAGGAC |
235 |
|
Exon 5 |
FH116 AGA AAA TCA ACA CAC TCT GTC CTG FH136 GGA AAA CCA GAT GGC CAG CG |
180 |
|
Exon 6 |
FH117 TCC TCC TTC CTC TCT CTG GC FH137 TCT GCA AGC CGC CTG CAC CG |
179 |
|
Exon 7 |
FH118 GGC GAA GGG ATG GGT AGG GG FH138 GTT GCC ATG TCA GGA AGC GC |
236 |
|
Exon 8 |
FH119 CAT TGG GGA AGA GCC TCC CC FH27 CCA CCC GCC GCC TTC CCG TGC TCA C |
197 |
|
Exon 9 |
FH9 TCC ATC GAC GGG TCC CCT CTG ACC C FH28 AGC CCT CAT CTC ACC TGC GGG CCA A |
273 |
|
Exon 10 (5’end) |
FH10 AGA TGA GGG CTC CTG GTG CGA TGC C FH29 GCC CTT GGT ATC CGC AAC AGA GAC A |
202 |
|
Exon 10 (3’end) |
FH11 GAT CCA CAG CAA CAT CTA CTG GAC C FH141 AGC CCT CAG CGT CGT GGA TA |
163 |
|
Exon 11 |
FH12 CAG CTA TTC TCT GTC CTC CCA CCA G FH142 GCT GGG ACG GCT GTC CTG CG* |
171 |
|
Exon 12 |
FH13 GCA CGT GAC CTC TCC TTA TCC ACT T FH32 CAC CTA AGT GCT TCG ATC TCG TAC G |
211 |
|
Exon 13 |
FH124 GTC ATC TTC CTT GCT GCC TG FH33 GTT TCC ACA AGG AGG TTT CAA GGT T |
218 |
|
Exon 14 |
FH265 GAA TCT TCT GGT ATA GCT GAT FH145 GCA GAG AGA GGC TCA GGA GG |
288 |
|
Exon 15 |
FH16 GAA GGG CCT GCA GGC ACG TGG CAC T FH146 GTG TGG TGG CGG GCC CAG TCT TT |
247 |
|
Exon 16 |
FH127 CCT TCC TTT AGA CCT GGG CC FH147 CAT AGC GGG AGG CTG TGA CC |
173 |
|
Exon 17 |
FH128 GGG TCT CTG GTC TCG GGC GC FH148 GGC TCT GGC TTT CTA GAG AGG G |
242 |
|
Exon 18 |
FH129 GCC TGT TTC CTG AGT GCT GG FH149 TCT CAG GAA GGG TTC TGG GC |
135 |
Oligonucleotides FH2-FH33 (Hobbs et al, 1992; Hum Mutat 1:445), FH51-52 (Gudnason et al, 1994); FH53-54 (Gudnason PhD thesis, 1995); FH55 & FH169 (Gudnason et al, 1993); FH110-149 (Leitersdorf et al, 1990; J Clin Invest 85:1014). (AAAGTCGAC) has a SalI site attached to the 5’end of FH51. * The first base in the 5’end is not on the gene map.
These combination of primers amplify under one condition, 1.5mMgCl2 and cycle conditions of: 96oC 5min x 1, (96oC 1min, 57oC 1min, 72oC 1min) x35, 72oC 5min (Day et al, 1997; Hum Mutat 10:116-27 & Heath et al, 1999; Atherosclerosis 143:41-54).
Table 4: Oligonucleotide sequences for the amplification of LDLR using DGGE (Lombardi et al, 1995; J Lipid Res 36:860).
|
Region/ exon |
Primer |
Nucleotide |
Oligonucleotide (Sense & antisense, 5’ to 3’) |
DMSO (10% (v/v) (+/-) & MgCl2 (mM) |
Denat grad (%) |
|
Exon 1 |
B2772 A2773 |
-255® 67+25 |
gAAT GCT GTA AAT GAC GTG G TTC TGG CGC CTG GAG CAA G |
- 1.5 |
60-80 |
|
Exon 2 |
B3118 A3119 |
68-25® 190+35 |
gCCT TTC TCC TTT TCC TCT CTC TC AAA ATA AAT GCA TAT CAT GCC CA |
+ 1.5 |
40-75 |
|
Exon 3 |
B3078 A3079 |
190-28® 313+26 |
gTGA CAG TTC AAT CCT GTC TCT TC AAT AGC AAA GGC AGG GCC ACA CT |
+ 1.5 |
40-75 |
|
Exon 4 |
A2534 B2535 |
314-25® 694+31 |
TGG TCT CGG CCA TCC ATC C gACG CCC CGC CCC CAC CCT G |
+ 1.0 |
50-80 |
|
Exon 5 |
A2536 B2537 |
695-26® 817+26 |
CAA CAC ACT CTG TCC TGTT* gGGG AAA ACC AGA TGG CCA GC |
+ 1.5 |
50-75 |
|
Exon 6 |
B2668 A2669 |
818-25® 940+25 |
gTCC TTC CTC TCT CTG GCA AGC CGC CTG CAC CGA G |
+ 1.5 |
40-70 |
|
Exon 7 |
B3120 A3121 |
941-25® 1060+24 |
gAGT CTG CAT CCC TGG CCC TGC GC AGG GCT CAG TCC ACC GGG GAA TC |
- 1.0 |
50-80 |
|
Exon 8 |
B2467 A2468 |
1061-24® 1186+25 |
gCCA AGC CTC TTT CTC CCA CCC GCC GCC TTC CCG T |
- 1.0 |
50-80 |
|
Exon 9 |
B1205 A1289 |
?® 1358+49 |
gGGC TGC AGG CAG GGG CGA CG CTG ACC TCG CTC CCC GGA CC |
+ 1.0 |
50-70 |
|
Exon 10 |
A2715 B2716 |
1359-25® 1705+29 |
ATG CCC TTC TCT CCT CCT G* gAGC CCT CAG CGT CGT GGA T |
+ 1.5 |
45-75 |
|
Exon 11 |
B2713 A2714 |
1587-25® 1705+29 |
gCAG CTA TTC TCT GTC TGGC TGG GAC GGC TGT CCT |
- 1.5 |
35-65 |
|
Exon 12 |
B2469 A2470 |
1706-25® 1845+24 |
gTCT CCT TAT CCA CTT TTC GAT CTC GTA CGT AAG |
- 1.5 |
35-75 |
|
Exon 13 |
B3122 A3123 |
1846-25® 1987+37 |
gGTC ATC TTC CTT GCT GCC TGT TT GTT TCC ACA AGG AGG TTT CAA GG |
- 1.5 |
35-70 |
|
Exon 14 |
B2717 A2718 |
1988-25® 2140+26 |
gCCT GAC TCC GCT TCT ACG CAG AAA CAA GGC GTG T |
- 1.5 |
40-70 |
|
Exon 15 |
A3124 B3125 |
2141-25® 2311+25 |
AGA AGA CGT TTA TTT ATT CTT TC gGTG TGG TGG CGG GCC CAG TCT TT |
- 1.5 |
40-80 |
|
Exon 16 |
B2770 A2771 |
2312-24® 2389+25 |
gCCT CAC TCT TGC TTC CGC TGG GGG ACC GGC CCG C |
- 1.5 |
55-80 |
|
Exon 17 |
B2768 A2769 |
2390-24® 2547+33 |
gTGA CAG AGC GTG CCT C TGG CTT TCT AGA GAG GGT C |
- 1.5 |
35-65 |
|
Exon 18 |
B2774 A2775 |
2548-24® 2627 |
gTCC GCT GTT TAC CAT T TCT CAG GAA GGG TTC TGG G |
- 1.5 |
50-80 |
GC Clamp (C2548) = g = CGC CCG CCG CGC GCC GCG CCC GTC CCG CCG CCG CC C GCC GCC GCC GCC CG.
The underline represents the 15bp GC-rich sequence that acts as a linker which is labelled as a g at the 5’end of either the sense or antisense primer. The 15bp GC-rich sequence was further elongated to a 50bp GC-sequence in a second PCR reaction, using primer C2548.* In reverse oligonucleotide, there is a G at 5’ end which is a T on the FH website gene map.
Two step amplification using the same cycle conditions for P1 and P2 (94oC 1min, 55oC 30s, 72oC 90s) x32, except for exons 9 and 12. Exon 12 has a P1 annealing temperature of 47oC, and there were completely different P1 cycle conditions for exon 9 (94oC 1min, 62oC 90sec, 72oC 2min) x34.
100% denaturant = 7M urea and 40% (v/v) deionised formamide.
Table 5: Oligonucleotide sequences for the amplification of LDLR using DGGE (Nissen et al, 1995; Hum Mutat 8:168).
|
Region/ exon |
Primer |
Nucleotide |
Oligonucleotide (Sense & antisense 5’ to 3’) |
Size (bp) |
Denat grad (%) |
|
Prom |
N1 N2 |
-255® -45 |
bAGG ACT GGA GTG GGA ATC AGA GC TGC TGT GTC CTA GCT GGA AAC CC |
252 |
30-70 |
|
Exon 1 |
N3 N4 |
-117® 67+23 |
aTTG AAA TGC TGT AAA TGA CGT GG CTG GCG CCT GGA GCA AGC |
256 |
40-80 |
|
Exon 2 |
N5 N6 |
68-57® 190+32 |
bCGT GGT CAG TTT CTG ATT CTG GCG ATA AAT GCA TAT CAT GCC CAA AGG |
253 |
30-70 |
|
Exon 3 |
N7 N8 |
191-55® 313+50 |
bTCG GCC TCA GTG GGT CTT TC ACT CCC CAG GAC TCA GAT AGG C |
268 |
30-70 |
|
Exon 4 (5’end) |
N9 N10 |
314-85® 484 |
bACT GCG GCA GCG TCC CCG GC GGA TGC AGG TGG AGC TGT TGC |
297 |
40-80 |
|
Exon 4 (3’end) |
N11 N12 |
439® 694+48 |
ACC TGT GGT CCC GCC AGC bCCA GGG ACA GGT GAT AGG ACG |
345 |
40-80 |
|
Exon 5 |
N13 N14 |
695-67® 817+48 |
bGGC CCT GCT TGT TTT TCT CTGG AGC AGC AAG GCA CAG AGA ATG G |
282 |
30-70 |
|
Exon 6 |
N15 N16 |
818-57® 940+36 |
bACG AAA CTG AGG CTC AGA CAC ACC GCT CCC CAC AAA CTC TGC AAG C |
262 |
30-70 |
|
Exon 7 |
N17 N18 |
941-34® 1060+56 |
bAGA GTG ACC AGT CTG CAT CCC TGG TTG GTT GCC ATG TCA GGA AGC |
253 |
40-80 |
|
Exon 8 |
N19 N20 |
1061-31® 1186+25 |
bTCC CCA CCA AGC CTC TTC TCT C CCA CCC GCC GCC TTC C |
222 |
40-80 |
|
Exon 9 |
N21 N22 |
1187-25® 1358+25 |
cCTG ACC TCG CTC CCC GGA CC GGC TGC AGG CAG GGG CGA CG |
278 |
40-80 |
|
Exon 10 |
N23 N24 |
1359-95® 1586+30 |
GCA GTG AGA TGA GGG CTC CTG G bCCT GCA GCC CTC AGC GTC G |
349 |
30-70 |
|
Exon 11 |
N25 N26 |
1587-51® 1705+29 |
bGGA TCC TCC CCC GCC CTC TGG CTG GGA CGG CTG TCC |
239 |
30-70 |
|
Exon 12 |
N27 N28 |
1706-64® 1845+92 |
GGC CCT CAG GCC CTC TGG bCCG AGT TTT CTG CGT TCA TCT T |
336 |
30-70 |
|
Exon 13 |
N29 N30 |
1846-25® 1987-46 |
aGTC ATC TTC CTT GCT GCC TG CAC AAG GAG GTT TCA AGG TTG G |
264 |
30-70 |
|
Exon 14 |
N31 N32 |
1988-37® 2140+34 |
aTCT CGT TCC TGC CCT GAC TCC GAC ACA GGA CGC AGA AAC AAG G |
274 |
40-80 |
|
Exon 15 |
N33 N34 |
2141-39® 2311+25 |
dGGC ACG TGG CAC TCA GAA GAC G GTG TGG TGG CGG GCC CAG TC |
288 |
40-80 |
|
Exon 16 |
N35 N36 |
2312-59® 2389+52 |
aCTC CAT TTC TTG GTG GCC TTC C CAT AGC GGG AGG CTG TGA CCT GG |
239 |
40-80 |
|
Exon 17 |
N37 N38 |
2390-36® 2547+35 |
aGGG CAG CTG TGT GAC AGA GCG CAT GGC TCT GGC TTT CTA GAG AGG |
279 |
30-70 |
|
Exon 18 |
N39 N40 |
2548-47® 2640 |
aCCT GAG TGC TGG ACT GAT AGT TTC C AAG GCC GGC GAG GTC TCA GG |
190 |
40-80 |
|
APOB |
N41 N42 |
3456® 35531 |
bGGA GCA GTT GAC CAC AAG CTT AGC GGT GGC TTT GCT TGT ATG TTC TCC |
382 |
30-70 |
a = 50bp GC Clamp = CGC CCG CCG CCG CCC GCC GCG CCC CGC GCC CGT CCC GCC GCC CCC GCC CG
b= 40bp GC Clamp = CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G
c= 46bp GC Clamp + 10bp AT Clamp =CGC CCG CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GAA ATA ATA AA
d= 3bp GC Clamp = CGG
5’ primer for the 5’ part of exon 4 (Jensen et al, 1994, Hum Mutat 4:102) and the 3’ primer for exon 9 (Top et al, 1993; Hum Genet 91:480). * Extra G at 5’ end which is not on the FH website gene map.
PCR conditions: All PCR’s contained 1.5mM MgCl2 except exon 4 (3’ end) - 1.0mM MgCl2 and exon 15 - 2.5mM MgCl2. Cycle conditions: (95oC 5min) x1, (94oC 1min, 66oC 5min) x40, (72oC 10min) x1, 99oC 7min, 65oC 60min, 37oC 60min, cooling to 4oC.
Table 6: Oligonucleotide sequences for the amplification of LDLR using DGGE (Ekstrom et al, 1998, E J Clin Investig 28:740).
|
Region/ exon |
Primer |
Nucleotide |
Oligonucleotide (Sense & antisense, 5’ to 3’) |
Size (bp) |
Denat grad (%) |
|
Promoter |
UE72 UE73 |
-276® -45 |
gCT GCC CTG GCG ACA CTT TCG AA TGC TGT GTC CTA GCT GGA AAC CC |
63 |
50-75 |
|
Exon 1 |
UE47 UE48 |
-120® 67+28 |
gCA CAT TGA AAT GCT GTA AAT GAC G CTA TTC TGG CGC CTG GAG CAA GCC |
63 |
60-80 |
|
Exon 2 |
UE87 UE81 |
68-57® 190+34 |
gCG TGG TCA GTT TCT GAT TCT GGC G AAA ATA AAT GCA TAT CAT GCC CAA AG |
60 |
50-75 |
|
Exon 3 |
UE05 UE06 |
191-27® 313+26 |
gTG ACA GTT CAA TCC TGT CTC TTC AAT AGC AAA GGC AGG GCC ACA CT |
63 |
50-75 |
|
Exon 4A |
UE80 UE49 |
314-24® 569 |
gTG GTC TCG GCC CAT CCA TCC CT* GGG GCT ACT GTC CCC TTG GA |
63 |
50-75 |
|
Exon 4B |
UE50 UE51 |
475® 694+48 |
gAC CTG CAT CCC CCA GCT GTG CCA GGG ACA GGT GAT AGG ACG |
63 |
60-80 |
|
Exon 5 |
UE09 UE10 |
695-25® 817+25 |
CAA CAC ACT CTG TCC TGT TT gGG AAA ACC AGA TGG CCA GCG |
52 |
50-75 |
|
Exon 6 |
UE11 UE12 |
818-27® 940+25 |
gTC CTT CCT CTC TCT GGC T GCA AGC CGC CTG CAC CGA G |
58 |
50-75 |
|
Exon 7 |
UE13 UE14 |
941-25® 1060+25 |
gAG TCT GCA TCC CTG GCC CTG CGC AGG GCT CAG TCC ACC GGG GAA TC |
63 |
50-75 |
|
Exon 8 |
UE15 UE16 |
1061-24® 1186+25 |
gCC AAG CCT CTT TCT CT CCA CCC GCC GCC TTC CCG T |
63 |
50-75 |
|
Exon 9 |
UE17 UE74 |
1187-25® 1358+25 |
CTG ACC TCG CTC CCC GGA CC gGG CTG CAG GCA GGG GCG ACG |
63 |
50-75 |
|
Exon 10 |
UE19 UE20 |
1359-25® 1586+25 |
ATG CCC TTC TCT CCT CCT G gAG CCC TCA GCG TCG TGG AT |
63 |
50-75 |
|
Exon 11 |
UE21 UE22 |
1587-25® 1705+29 |
gCA GCT ATT CTC TGT C TGG CTG GGA CGG CTG TCC T |
66 |
50-75 |
|
Exon 12 |
UE23 UE24 |
1706-25® 1845+35 |
gTC TCC TTA TCC ACT TGT GTG CTT CGA TCT CGT ACG TAA GCC AC |
58 |
50-75 |
|
Exon 13 |
UE25 UE26 |
1846-25® 1987+51 |
gGT CAT CTT CCT TGC TGC CTG TTT GTT TCC ACA AGG AGG TTT CAA GG |
63 |
40-65 |
|
Exon 14 |
UE27 UE28 |
1988-24® 2140+26 |
gCC TGA CTC CGC TTC T ACG CAG AAA CAA GGC GTG T |
60 |
60-80 |
|
Exon 15 |
UE29 UE30 |
2141-21® 2311+25 |
AGA AGA CGT TTA TTT ATT CTT TC gGT GTG GTG GCG GGC CCA GTC TTT |
53 |
50-75 |
|
Exon 16 |
UE31 UE32 |
2312-25® 2389+25 |
gCC TCA CTC TTG CTT CTC TCC CGC TGG GGG ACC GGC CCG C |
63 |
50-75 |
|
Exon 17 |
UE76 UE77 |
2390-36® 2547+35 |
GGG CAG CTG TGT GAC AGA GC gCA TGG CTC TGG CTT TCT AGA G |
63 |
40-65 |
|
Exon 18 |
UE79 UE71 |
2548-25® 2644 |
gTC CGC TGT TTA CCA TTT GTT G ACA AGG CCG GCG AGG TCT |
60 |
60-80 |
|
APOB |
UE68 UE65 |
gGG AGC AGT TGA CCA CAA GCT TAG C GGT GGC TTT GCT TGT ATG TTC TCC |
66 |
30-55 |
GC Clamp = g = GCG CCG CGC CCG TCC CGC CGC CGC CCG CCG CCG GCG CCC G
* Extra C in UE80 sequence compared to FH website gene map. **
Cycle conditions: (95oC 1min, 72oC 2min) x32, (95oC 5min, 72oC 6min) x1.
100% denaturant = 7mM urea, 40% (v/v) deionised formamide
Table 7: Oligonucleotide sequences for sequencing PCR’s and sequencing reactions (Heath et al, unpublished).
|
Region/ exon |
Oligonucleotide sequences (Sense & antisense, 5’ to 3’) |
Size (bp) |
|
Promoter & exon 1 |
FHA GTC AGC TCT TCA CCG GAG ACC CAA ATA CA FH131 (Table 2) |
414 |
|
Exon 3 |
FH4 (Table 1) FH23 (Table 1) |
180 |
|
Exon 4 |
Ex4F CGG CTA TAG AAT GGG CTG GTG TTG Ex4R TGT TGT TGG AAA TCC ACT TCG GCA |
557 |
|
Exon 10 |
FH340 TTG GCC CGC AGT GAG ATG AGG FH141 (Table 2) |
310 |
|
Exon 14 |
FH342 CTT CCA CAA CCT CAC CCA GCC A FH145 (Table 2) |
380 |
Oligonucleotides FH 4, FH23 (Hobbs et al, 1992, Hum Mutat 1:445) and FH131, FH141, FH145 (Leitersdorf et al,1990, J Clin Invest 85:1014).
These combination of primers amplify under one condition, 1.5mMgCl2 and cycle conditions of: 96oC 5min x 1, (96oC 1min, 57oC 1min, 72oC 1min) x35, 72oC 5min (Day et al, 1997; Hum Mutat 10:116-27 & Heath et al, 1999; Atherosclerosis 143:41-54).