Document Sample
1967 Powered By Docstoc
					cence light microscopy. J Cell Biol 1987;105:41-8.                                                                      nique of glycoprotein                   lectin   immunosorbent              assay   (GLIA).   Biol
23. Ploem JS. New instrumentation     for sensitive                                            image analysis           Chem Hoppe                Seyler      1988;369:1157-66.
of fluorescence                  in cells         and      tissues.        In: Tayer       DL, Waggoner     AS,         27. Kinoshita                  N, Suzuki 5, Matsuda Y, Taniguchi                   N. a-Fetopro-
Lanni F, Murphy R, Birge R, eds. Applications                   of fluorescence       in                                tein         antibody-lectin          enzyme       immunoassay        to    characterise     sugar
the biological      sciences.  New York: Alan R Liss, 1986;289-300.
                                                                                                                        chains            for     the      study        of liver diseases.          Clin     Chim     Acta
24. Fodor SPA, Read JL, Pirrung                     MC, et al. Light-directed,
spatially      addressable      parallel       chemical     synthesis.        Science
1991;251:767-73.                                                                                                        28. Shalev       V, Greenberg        GH, McAlpine        PJ. Detection      of at-
25. Ekins RP. Immunoassay             standardization.     In: Kallner A, Magid                                         tograms of antigen by a high sensitivity             enzyme-linked      immuno-
E, Albert W, eds. Improvement             of comparability      and compatibility                                       sorbent   assay (HS-ELISA)       using a fluorogemc      substrate.  J Immunol
of laboratory     assay results in life sciences. Immunoassay              standard-                                    Methods     1980;38:125.
ization.   Scand J Clin Lab Invest 1991;51(Suppl              205):33-46.                                               29. Harris       CC, Yolken RH, Kroken            H, Hsu IC. Ultrasensitive
26. Kottgen                   E, Hell       B, Muller                 C, Tauber        R. Demonstration           of    enzymatic      radioimmunoassay:        application to detection       of cholera
glycosylation                  variants      of human                  fibrinogen,      using the new         tech-     toxin and rotavirus.        Proc Nat! Aced Sci USA 1979;76:5336.

Vol 37, pp. 1447-8:      In our desire for rapid publication,
important   errors     were introduced       into the following
Technical   Brief. The corrected       version     is here repro-
duced in its entirety,     with our apologies     to the authors.

Rapid Detection         of 1717-1G--*A           Mutation In CFTR Gene
by PCR-Mediated            Site-Directed         Mutagenesis,      Laura
Cremonesi,1      Manuela        Seia,2 Carmelina         Magnani,1     and                                              Fig.     1. Detection             of the 1717-1 G-.A       mutation        by PCR
Maurizio    Ferrari1      (1 Istituto      ScientificoH.S. Raffaele,                                                    Reactions   were carried out with 1 tg of genomic DNA in a total volume of 100
                                                                                                                        L containing 10 mmol/L Tris HCI (pH 8.3), 50 mmoL/L KCI, 1.5 mmovL
Lab. Centrale,       Milano;       2 Istituti   Clin. di Perfezionamento,                                               MgCI2, 0.1 9/L gelatin, 200 MInOl/L each of the four deoxynbonucleotide
Lab. di Ricerche        Clin., Milano,        Italy)                                                                    tnphosphates,    2.5 units of Taq polymerase      (Perkin-Elmer Celus, Norwalk,
                                                                                                                        CT), and 100 pmol of each of the primers. PCR conditions were as follows:
       Until           now,      among           the non-AF508                   mutations       identified       in    denaturation at 94 #{176}C 1 mm, annealing at 55 #{176}C a, and extension at
                                                                                                                                                                                for 30
 the       cystic         fibrosis          transmembrane                        conductance       regulator            72#{176}C 1 mm, for a total of 30 cycles. PCR products
                                                                                                                               for                                                 were digested   for 2 hat
                                                                                                                        37#{176}C 5 U of AvaIl and electrophoresed       on 3% agarose-1%       NuSieve gel
 (CVFR)                by the Cystic Fibrosis
                       gene                                                       (CF) Genetic      Analysis            for 1 h at 50 V. Bands were made visible by staining the gel with ethidium
 Consortium,          the ones most frequently                                       seen    in our popula-             bromide. Lane 1: HaeIIl-digested        pBR322    size marker.    Lane 2: normal
 tion sample are the 1717-1G--A mutation (13/144 or 9% of                                                               homozygote. Lane 3: CF patient homozygous for the 1717-1G-’A mutation.
                                                                                                                        Lane 4: heterozygote carrier    for the 1717-1G-A     mutation
 the CF chromosomes)               and the G542X         mutation     (16/190      or
 8.4% of the CF chromosomes),                     both revealed     by dot-blot
 hybridization          of the polymerase         chain reaction (PCR) prod-
 uct with allele-specific           oligonucleotides       (ASO) probes       (1).
     In an attempt            to simplify        the analysis     of the most                                                  For      the     forward       primer,      we used the one made available
 frequent      mutations        in the CFFR        gene, we converted       radio-                                      by the CF Genetic                      Analysis     Consortium  to amplify exon 11
 labeled ASO detection    into restriction                                             endonuclease           anal-     of the   CFFR                        gene:      5’-CAACTGTGGrFAAAGCAAT-
 ysis of the amplified product.                                                                                         AGTGT-3’.
                                                                                                                            Digestion       by A vail enzyme           of the PCR product         generates
     A PCR-mediated                              site-directed               mutagenesis          (2, 3) to de-
                                                                                                                        two fragments            of 116- and 21-hp in the wild-type                     alleles
 tect the G542X                      mutation                  by generating             a novel BstNi          site
                                                                                                                        and leaves          undigested        a 137-hp       fragment      in the mutant
 in the wild-type                     sequence                  had already            been suggested           (4).
                                                                                                                        alleles     (Figure      1).
     To detect the 1717-1G-A             mutation, we designed     the
                                                                                                                            By combined           analysis      for the F508          mutation       (5) (252/
 reverse primer (5’-CTCTGCAAACITGGAGAGGTC-3’)                        to
                                                                                                                        470 or 53.6%           of the CF chromosomes),                 1717-1G---A,          and
 contain  a single-base        mismatch    (T-’G), which could create
                                                                                                                        G542X,         about      71% of mutations              might     be detected           by
 a novel Avail     restriction      site [G     G(A/T)CC]  in the am-
                                                                                                                        nonisotopic         analysis       of the PCR product,           thus    allowing         a
 plified       wild-type                  (WT)       allele       but       not in the        CF mutant         (M)
                                                                                                                        faster     and easier        one-day       procedure      for carrier      screening
                                                                                                                        and prenatal          testing.

 WT:            WT                   1717
                                                                                                                        1. Kerem      B, Zielenski     J, Markiewicz      D, et al. Identification      of
                                  TAGGACA                                  GCAGAG#{176}                                 mutations     in regions   corresponding     to the two putative     nucleotide
                                                                                                                        (ATP)-binding      folds of the cystic fibrosis gene. Proc Nat! Aced Sci
                              3ATCTG..J                                    CGTCTC5,                                     USA 1990;87:8447-51.
                                                                                                                        2. Haliassos        A, Chomel JC, Baudis M, Kruh J, Kaplan JC, Kit.zis
                                      Avail             site                                                            A. Modification         of enzymatically         amplified     DNA for the detection
                                                                                                                        of point mutations.          Nucleic Acids Res 1989;17:3606.
M:              M                  1717                                                                                 3. Friedman          WE, Highsmith         E Jr, Prior TW, Perry TR, Silverman
                                                                                                                        LM. Cystic fibrosis deletion              mutation       detected     by PCR-mediated
                                                                                                                        site-directed       mutagenesis       [Tech Brief]. Clin Chem 1990;36:695-6.
                         5’TAAGACA                                       GCAGAG3                                        4. Ng ISL, Pace R, Richard MV, et a!. Methods for analysis of
                                                                                                                        multiple      cystic fibrosis mutations.           Hum Genet (in press).
                                                                                                                        5. Ferrari      M, Cremonesi         L. More on detection           of cystic fibrosis by
                                A1TCTGQ                                 CGTCTC                                          polymerase          chain reaction         [Response        to    Letter].   Clin Chem
                                                                                                 5,                     1990;36:1702-3.
                ‘1’,    mutagenized                     base     of reverse          primer

                                                                                                                                                 CLINICAL          CHEMISTRY,       Vol. 37, No. 11, 1991             1967

Shared By: