Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA.
We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5' or 3' splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons.
Fuentes
J J
JJ
Medical and Molecular Genetics Center-IRO, L'Hospitalet de Llobregat, Barcelona, Spain.
Pucharcós
C
C
Pritchard
M
M
Estivill
X
X
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004314:Down Syndrome / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D012326:RNA Splicing; D012091:Repetitive Sequences, Nucleic Acid; D012641:Selection, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D011506:Proteins; C108612:RCAN1 protein, human