Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3' --> 5' exonuclease activity of DNA polymerases on 3'-terminal isoheptyl amino groups.
Numerous steps are required to prepare a sequencing library for serial analysis of gene expression (or SAGE) from an original mRNA sample. The presence of inefficiencies, however, can lead to a cumulative loss of sample during processing which can yield a library of short sequence tags (SSTs) that represents only a minute fraction of the original starting sample, potentially compromising the quality of the analysis and necessitating relatively large amounts of starting material. We show here that commonly observed higher molecular weight (HMW) amplification products observed following the PCR amplification of ditags are a direct result of the presence of HMW ligation products created during ditag formation. Using model tags, we demonstrate that the formation of these HMW ligation products becomes permissible following the release of the 3'-terminal isoheptyl amine (3'-IHA) from the SST during the fill-in reaction with the Klenow fragment (KF) of DNA polymerase (DNAP) I and is mediated by its 3' --> 5' exonuclease activity. We further show that the incorporation of SSTs into HMW ligation products can lead to a loss of sequence information from SAGE analysis, potentially skewing sequencing results away from the true distribution in the original sample. By modifying fill-in conditions through the use of Vent DNAP at 12 degrees C and by including terminal phosphorothioate linkages within the SAGE adaptors to specifically inhibit exonucleolytic removal of the 3'-terminal amine, we are able to maximize the yield of ditags and bypass the need for gel purification via PAGE following PCR. The modifications described here, combined with the modifications described previously by our group for adaptor ligation, ensure that the full sequence information content in SSTs derived from the transcriptome is preserved in the pool of amplified ditags prior to the creation of a SAGE library.
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z3.
Austin P
AP
So
Robin F B
RF
Turner
Charles A
CA
Haynes
D020413:3' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000737:chemistry; D001483:Base Sequence; D003198:Computer Simulation; D003455:Cryptococcus neoformans / Q000737:chemistry / Q000254:growth & development; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D020224:Expressed Sequence Tags; D015723:Gene Library; D021141:Nucleic Acid Amplification Techniques; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D009841:Oligonucleotides; D012331:RNA, Fungal; D012333:RNA, Messenger; C070331:Tli polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases