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Table 1 PCR primers and conditions for amplifications of PKD1 cDNA and DNA

From: Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

Primer

Primer Sequence (5'->3')

Nucleotide Position

Location in PKD1Transcript

PCR Product Size (bp)

Annealing Temperature (°C)

Mg2+ Conc. (mM)

 

cDNA amplification

     

TH1F

CTGGGGACGGCGGGGCCATGCG

175–196 a

5' UTR

13,634

68

1.2

TH1B

GGCCTGGGGCAAGGGAGGATGACAA

13808–13784 a

3' UTR

   

SI1F

ACGGCGGGGCCATGCGCGCGCTGCCCTAAC

181–210a

5'UTR

1,554

72

3.0

SI1B

CAGTGCTCGGCTGTGGCTGGGTGTGGCTCC

1734–1705a

Exon 7

   

SI2F

AGGAGCCTAGACGTGTGGATCG

1595–1616a

Exon 6–7

1,678

65

1.5

SI2B

CCTGCATCCTGTTCATCCGCTC

3272–3251a

Exon 13

   

SI3F

CATCAACGACAAGCAGTCCCT

3115–3135a

Exon 12

1,575

60

1.5

SI3B

CCACGGCCCACAGCAGAGAA

4689–4670a

Exon 15

   

SI4F

ATCTCTGCTGCCAATGACTCAG

4562–4583a

Exon 15

1,473

60

1.5

SI4B

GGGGAAGCTGTGGGAGAAAC

6034–6015a

Exon 15

   

SI5F

TCAATGCCTCCAACGCAGTCAGC

5808–5830a

Exon 15

1,663

65

1.5

SI5B

GTCTCATCCAGCACCAGCGTCTTG

7470–7447a

Exon 18

   

SI6F

AGCAGCGGCTCCAAGCGAG

7400–7418a

Exon 17

1,506

65

3.0

SI6R

CACAACGGAGTTGGCGGAGT

8905–8886a

Exon 23

   

SI7F

CCTTTCCCTTTGGCTATATCAG

8709–8730a

Exon 23

1,518

60

1.5

SI7B

CCAGGTAGACGGGATAGACAAC

10226–10205a

Exon 30

   

SI8F

GCCACCTGCTGCGTTCTCCT

10052–10071a

Exon 29

1,539

65

1.5

SI8B

CGTCCCCGAGCCATTGTGAG

11590–11571a

Exon 40

   

SI9F

CTTCAGCACCAGCGATTACGACGTT

11533–11557a

Exon 40

1,650

65

1.5

SI9B

AGAAAGTAATACTGAGCGGTGTCCACTC

13182–13155a

3' UTR

   
 

Genomic DNA amplification

     

SI3.1F

GCAACGTCACCGTGAACTACAACGTAACCG

26340–26369 b

Exon 13

18,099

61

1.0

Ex34B

GAGCAGGTCCGTTTCCATGTGGGTGTCTTG

44438–44409 b

Exon 34

   

SI4.2F

CTTCCCCACCAACCACACGGTACAGC

29064–29039

Exon 15

486

62

1.0

SI4.2B

AGGCCACTCACAGGCACCTGCACATC'

29523–29498

Exon 15

   

SI4.1F

AGCCAACGCCACCGTGGAA

29476–29494b

Exon 15

372

60

1.5

SI4.1B

GCAGCCAGCAGGATCTGAAAATG

29847–29825 b

Exon 15

   

SI6.1F

CGCTGTGCACGCCCTCACCACCAAGGT

32835–32861 b

Exon 18

480

60

1.5

SI6.1B

GTGCAGCCAGACTGTGAGCCCCGTTGC

33314–33288 b

Exon 20

   

SI7.2F

GGAGTTACCATCTGAACCTCTCCAG

38978–39002 b

Exon 25

698

60

1.0

SI7.2B

CAGGATGAACACACGAGCCCTTCACAC

39675–39649 b

IVS 26

   

SI8L

TTCTTTGACAAGCACATCTGGC

41600–41579

Exon 29

336

57

1.0

SI7B

CCAGGTAGACGGGATAGACAAC

41915–41894

Exon 30

   
  1. aThe nucleotide positions are according to HUMPKD1A, GenBank Accession No. L33243. bThe nucleotide positions are according to HUMPKD1GEN, GenBank Accession No. L39891. Bold faces are primers for long-range PCR of which products were used as PKD1-specific templates for nested PCRs.