An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.
A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.
Borodin
A
A
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow.
Kopatnzev
E
E
Wagner
L
L
Volik
S
S
Ermolaeva
O
O
Lebedev
Y
Y
Monastyrskaya
G
G
Kunz
J
J
Grzeschik
K H
KH
Sverdlov
E
E
D000818:Animals; D001483:Base Sequence; D002888:Chromosomes, Human, Pair 19; D002892:Chromosomes, Human, Pair 22; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D014960:X Chromosome
D017931:DNA Primers; D018076:DNA, Complementary; D012332:RNA, Heterogeneous Nuclear