High-performance affinity chromatography of messenger RNA.
A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry. The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces. Oligomers which differed in length by a single nucleotide base were readily resolved. Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min. The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions. The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length. While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths. Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed. These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.
Department of Biology, Purdue University School of Science, Indianapolis, IN 46205.
T A
TA
Goss
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M
Bard
H W
HW
Jarrett
D011232:Chemical Precipitation; D002712:Chlorides; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008094:Lithium; D018021:Lithium Chloride; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D011071:Poly T; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012331:RNA, Fungal / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012822:Silicon Dioxide
D002712:Chlorides; D012331:RNA, Fungal; D012333:RNA, Messenger; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D012822:Silicon Dioxide; D008094:Lithium; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D011130:RNA Ligase (ATP); D018021:Lithium Chloride