PCR based targeted genomic and cDNA differential display.
We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.
Broude
N E
NE
Center for Advanced Biotechnology, Boston University, MA 02215, USA.
Storm
N
N
Malpel
S
S
Graber
J H
JH
Lukyanov
S
S
Sverdlov
E
E
Smith
C L
CL
D000483:Alleles; D018076:DNA, Complementary; D006801:Humans; D006816:Huntington Disease / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D018911:Trinucleotide Repeats
D018076:DNA, Complementary