TY - JOUR

T1 - DNA sequence analysis by MALDI mass spectrometry

AU - Kirpekar, F

AU - Nordhoff, E

AU - Larsen, L K

AU - Kristiansen, K

AU - Roepstorff, P

AU - Hillenkamp, F

N1 - Keywords: Animals; Humans; Mice; Receptors, Cytoplasmic and Nuclear; Receptors, LDL; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription Factors; alpha 1-Antitrypsin

PY - 1998

Y1 - 1998

N2 - Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.

AB - Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.

M3 - Journal article

C2 - 9592136

VL - 26

SP - 2554

EP - 2559

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 11

ER -