Double-stranded DNA analysis by matrix assisted laser desorption/ionization

Conference · Sat Dec 31 04:00:00 EST 1994

OSTI ID:61957

Tang, K ^[1]; Taranenko, N I; Allman, S L; Chen, C H ^[2]; Chang, L Y ^[3]

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1. Vanderbilt Univ., Nashville, TN (United States)
2. Oak Ridge National Lab., TN (United States)
3. Univ. of Tennessee Medical Center, Knoxville, TN (United States)

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry was used to analyze double-stranded DNA fragments digested with restricted enzymes. As previously reported, only denatured single strands were detected and the resolution of TOFMS was not good enough to distinguish the mass difference between the complementary strands. To prove denaturation occurred rather than detecting doubly charged ds-DNA fragments, new DNA segments and restriction enzyme cleavage sites were searched. A 246 bp DNA designated as pLB129 was used. This segment contains part of the human immunoglobulin V{sub {lambda}} germline gene. It has several restriction sites that provide fragments with sticky ends. The 246 bp DNA was amplified by polyermase chain reaction (PCR), and digested with three kinds of restriction enzymes. The final products were purified with phenol/chloroform extraction and alcohol precipitation. MALDI-MS results clearly indicated that denaturation did happen as the mass peak corresponding to each individual was registered. 3-hydroxypicolinic acid (3-HPA) was used as the matrix. Analysis of double stranded DNA by MALDI-MS will have significant impacts in molecular biology. As coupled with PCR technique, MALDI-MS has great potential in fast screening tests for biomedical applications.

• DOE Contract Number: AC05-84OR21400
• OSTI ID: 61957
• Report Number(s): CONF-9405234--
• Country of Publication: United States
• Language: English

Related Subjects

40 CHEMISTRY
55 BIOLOGY AND MEDICINE
BASIC STUDIES
DESORPTION
DNA
MASS SPECTROSCOPY
SCREENING
TIME-OF-FLIGHT MASS SPECTROMETERS