The Toniolo Lab at San Raffaele, Home page | The Toniolo Lab at San Raffaele, 'supplementary Data' page

Mutation analisis of LMNA and emerin; Vytopil et al., 2002

LMNA
Emerin

LMNA primers+PCR conditions

EXON

LENGTH (bp)

PRIMER F

PRIMER R

HOT START

DENAT

ANNEAL

ELONG

fin. ext.

cycles

exon 1a

350

LA1F

LMNA 1aR2

96° 5 '

96° 1'

61° 45''

72° 1'

72° 7'

35x

 

 

 

 

 

 

 

 

 

 

exon 1b

332

LMNA1bF2

LMNA1bR

96° 5 '

96° 1'

61° 45''

72° 1'

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 2

339

LA2FB

LA2RF

96° 5'

96° 1'

57° 30''

72° 1'

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 3

232

LMNA3F2

LMNA3R2

96° 5'

96° 1'

61° 1'

72° 1'

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 4

341

LMNA4F

LA4R

96° 5'

96° 1'

62°45''

72° 1'

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 5

231

LMNA5F

LMNA5R

96° 5'

96° 1'

61° 1'

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 6

376

LA6F

LA6R

96° 5'

96° 1'

61° 1'

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 7

359

LA7F

LA7R

96° 5'

96° 1'

63° 45''

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 8

205

LA8F

LA8R

96° 5'

96° 1'

61° 45''

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 9

239

LA9F

LA9R

96° 5'

96° 1'

61° 1'

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 10

319

LMNA10F

LMNA10R

96° 5'

96° 1'

59° 1'

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 10B

214

LA10FB

LA10RB

96

96° 1'

62° 45''

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 11

359

LMNA11F

LMNA11Rev

96° 5'

96° 1'

59° 1'

72° 1

72° 7'

40x

 

 

 

 

 

 

 

 

 

 

exon 12

198

LMNA12F

LMNA12R

96° 5'

96° 1'

61° 45'

72° 1

72° 7'

40x

index of tables




LMNA primers

Exon

Forward primer

Reverse primer

 

1A

LA1F/LMNA 1aR2

CCC AgA TCC CgA ggT CCg AC

AgC CCT gCg TTC TCC gTT TCC

1B

LMNA1bF2/LMNA1bR

ACC CgC ATC ACC Cgg CTg

CCT CTC CAC TCC CCg CCA

 

 

 

 

2

LA2FB/LA2RB

ATT gCA ggC ATA gCA gCg C

gAg ggC CTA ggT AgA AgA G

 

 

 

 

3

LMNA3F2/LMNA3R2

TTC TTg TgT TCT gTg ACC CCT T

CCC AAg TCT gTC ATC ACC CA

 

 

 

 

4

LMNA4F/LA4R

gTC CCT ggg TCT Tgg CCT CC

gCC ACC ATC TgC CTg ATC C

 

 

 

 

5

LMNA5F/LMNA5R

CCT CCA CCC CTC CCA gTC AC

TgC ATC Cgg CCC AgA CTC Tag

 

 

 

 

6

LA6F/LA6R

Cag CTg TCT CCT ACA CCg ACC

ggT CTA gTC AAg gCC AgT TgC

 

 

 

 

7

LA7F/LA7R

gAg gTg CTg gCA gTg TCC TC

CTT CTg TCT TgC CAC TCT CTC C

 

 

 

 

8

LA8F/LA8R

ACC CAA gAg CCT ggg TgA gC

CAC CCA Agg TCT CCC CAg Ag

 

 

 

 

9

LA9F/LA9R

TTg ggT gTC gAT ggg AgC gC

gCA gCT gCC TCC gAT gTT gg

 

 

 

 

10

LMNA10F/LMNA10R

ACC CTT CCC Tgg CCC TgA C

CAC CTg ggT TCC CTg TTC AAg

 

 

 

 

10B

LA10FB/LA10RB

gCA AgC Tgg TgC gCT Cag Tg

ggC CAg gCC AgC gAg TAA Ag

 

 

 

 

11

LMNA11F/LMNA11Rev

ggT CAg TCC CAg ACT CgC C

CgC CTg CAg gAT TTg gAA gAC

 

 

 

 

12

LMNA12F/LMNA11R

gTg Agg gAT ggg ggA gAT gC

ggg Tgg gCA TgA ggT gAg g

index of tables




LMNA DHPLC conditions

EXON

Temperature of analysis °C

Gradient of acetonitrile (%)

1A

66

51-56-64

 

68

49-54-62

1B

65

52-57-65

 

66

51-56-64

2

62

51-56-64

 

64

49-54-62

3

61

48-53-61

 

63

46-51-59

4B

63

50-55-63

 

66

45-50-58

5

65

48-53-61

6

65

50-55-63

7

64

52-57-65

 

65

51-56-64

8

62

48-53-61

 

64

45-50-58

9

64

49-54-63

10

65

48-53-61

10B

64

46-51-59

 

66

45-50-58

11

65

52-57-65

 

67

48-53-61

12

62

47-52-60

 

63

46-51-59

index of tables




Emerin primers+PCR conditions

EXON

primers

HOT START

DENAT

ANNEAL

ELONG

fin. ext.

n°cycles

Cycler

1

1NF/1R

96° 5 '

96°30''

64°45''

72°45''

72° 7'

40x

9600

 

 

 

 

 

 

 

 

 

2

2F/2NR

96° 5 '

96° 30''

61° 30''

72° 30''

72° 7'

40x

9700

 

 

 

 

 

 

 

 

 

3

3NF/3NR

96° 5 '

96° 30'

63° 30''

72° 30''

72° 7'

35

9700

 

 

 

 

 

 

 

 

 

4

4F/4R

96° 5'

96° 1'

66°1'

72° 1'

72° 7'

40x

9600

 

 

 

 

 

 

 

 

 

5

5AF/6AR

96° 5'

96° 1'

67°1'

72° 1'

72° 7'

40x

9700

 

 

 

 

 

 

 

 

 

6A

6AF/6AR

96° 5'

96° 30'

64° 30''

72° 30''

72° 7'

35

9700

 

 

 

 

 

 

 

 

 

6B

6BF/6NBR

96° 5'

96° 1'

61°1'

72° 1'

72° 7'

40x

9600


index of tables



Emerin primers

Forward

Reverse

 

1NF/1R

GCCGCTCCCAATGCGCTCG

CCCTCCTCGTGCTCCGCCT

 

 

 

2F/2NR

TCGTGCTCCGCCTCGCGAC

GGATGGGGTCGCGAGGGTG

 

 

 

3NF/3NR

GGTCGAGAGCGGCACTGGAG

GACCCCGCTCACAGGGAGGA

 

 

 

4F/4R

CAGGCGGGGCACACCGATGC

CCTAGGGGATCGCGGCTGTGT

 

 

 

5AF/6AR

CCCTGCCAGCCAGTCCCCTC

CATGGCTCACCCGCCGTGCC

 

 

 

6AF/6AR

CCAGGGACGGGCTGGTTCTG

CATGGCTCACCCGCCGTGCC

 

 

 

6BF/6NBR

AGAGCATCACGCACTACCGC

CTTCAGAGCTAGGTCTTTGGGG

index of tables




Emerin DHPLC conditions

Exon

Analysis temperature

Gradient B [%]

1

67

48-53-61

 

69

47-52-69

 

 

 

2

64

50-55-63

 

66

48-53-61

 

 

 

3

60

45-50-58

 

61

47-52-60

 

 

 

4

63

48-53-61

 

64

47-52-60

 

 

 

5

60

51-56-64

 

61

51-56-64

 

 

 

6A

64

44-49-67

 

 

 

6B

62

49-54-62

 

65

43-48-56

 

index of tables

The Toniolo Lab at San Raffaele, Home page | The Toniolo Lab at San Raffaele, 'supplementary Data' page