Format

Send to

Choose Destination
Biotechniques. 2009 Jan;46(1):51-2, 54-7.

Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.

Author information

1
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. tomislav_maricic@eva.mpg.de

Abstract

To increase the yield of DNA sequence generated by the 454 technology from small amounts of starting DNA, we investigated the efficiency of each step in the 454 library preparation process. We find that the last step, when the single-stranded library is released by NaOH, is inefficient and highly variable. When this step is replaced with heat treatment, library amounts dramatically increase. Furthermore, when sequencing templates are first isolated by NaOH treatment and subsequently by heat treatment, the sequences of both strands of individual template DNA molecules can be determined. Using this approach, we confirm that C/G base pairs observed as T/A base pairs in Neanderthal DNA sequences are due to a modification of the cytosine rather than guanine residues.

PMID:
19301622
DOI:
10.2144/000113042
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Atypon
Loading ...
Support Center