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Flanking region sequences and internal repeat structure of the pYNH24 (D2S44) 2 kbp insert analyzed by polymerase chain reaction and partial digestion with Rsa I
Author(s) -
Holmlund Gunilla,
Lindblom Bertil
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601308
Subject(s) - tandem repeat , genetics , restriction enzyme , insert (composites) , biology , restriction site , polymerase chain reaction , primer (cosmetics) , repeated sequence , restriction fragment , dna , microbiology and biotechnology , gene , genome , chemistry , mechanical engineering , organic chemistry , engineering
The 2 kbp YNH24 pUC18 insert was partially sequenced, amplified with fluorescence‐labeled primers, and characterized by incomplete digestion with the restriction enzyme Rsa I. A characteristic Rsa I profile with one restriction site in each repeat unit was obtained when the fragments were analyzed on a DNA sequencer. We developed this procedure with the specific aim of analyzing the internal repeat structure in recombinant alleles often observed in the YNH24 variable number of tandem repeat system (D2S44). Nonetheless, it should also be possible to apply the method when analyzing other tandem repeats, even when little information regarding their sequences is available. In addition, when the repeat array is amplified, either a 5′ or a 3′ fluorescence‐labeled primer can be used to enable analysis from both directions of fairly long (> 3.6 kbp) alleles. The Rsa I fragments obtained by partial digestion of the amplified pYNH24 insert corresponded well to the Rsa I sites found in the sequenced regions. The 5′ flanking region contained eight 30–34 bp sequences similar to the repeat unit and also several Rsa I sites, whereas only a few Rsa I sites and no repeat units were found in the 3′ flanking region. Twenty‐five consecutive Rsa I sites were revealed, in the center of the pYNH24 insert, indicating a region with strictly repeated core sequences; this repetitive block comprised less than half of the total insert. Two approximately 2.24 kbp Hin fI alleles from two different heterozygous individuals were found to have identical internal structure, which was very similar to that of the insert although it exhibited only 15 Rsa I repeats.