Cloning the shared components of complex DNA resources.
The complex and repetitive nature of mammalian genomes limits the ability of conventional molecular techniques to recover sequences of interest. Here we describe a rapid and simple procedure for the direct cloning of sequences which are coincident between DNA mixtures of whole genome complexity. The system, called end ligation coincident sequence cloning (EL-CSC), can enrich coincident DNA by greater than 10(6)-fold and overcomes problems associated with repetitive elements. Applying EL-CSC to various paired DNA resources enables the facile cloning of both genomic markers and novel genes. To demonstrate the power of the method we have i) selectively purified single copy sequences from a complete genome, and ii) isolated gene fragments from 260 kb of cloned genomic DNA.
Brookes
A J
AJ
Molecular Genetics Section, MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK.
Slorach
E M
EM
Morrison
K E
KE
Qureshi
S J
SJ
Blake
D
D
Davies
K
K
Porteous
D J
DJ
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005819:Genetic Markers; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D005819:Genetic Markers