Manufacturing DNA microarrays from unpurified PCR products.
For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.
Diehl
Frank
F
Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, 69120 Heidelberg, Germany.
Beckmann
Boris
B
Kellner
Nadine
N
Hauser
Nicole C
NC
Diehl
Susanne
S
Hoheisel
Jörg D
JD
D000818:Animals; D002176:Candida albicans / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003890:Desiccation; D004331:Drosophila melanogaster / Q000235:genetics; D005456:Fluorescent Dyes; D015723:Gene Library; D005898:Glass; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000295:instrumentation* / Q000379:methods*; D016366:Open Reading Frames / Q000235:genetics; D011107:Polylysine; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000235:genetics; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D012821:Silanes; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000235:genetics
D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012821:Silanes; D011107:Polylysine; D004247:DNA