Solid-phase sequencing

B5. Berg, C., Hedrum, A., Holmberg, A., Ponten, F., Uhler, M. et al., Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides. Clin. Chem. (Winston-Salem, N.C.) 41, 1461-1466 (1995). 

Hultman, T., Stahl, S., Hornes, E., and Chlen, M. (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17,4937—4946. 

This category represents the most popular subclass of cyclative cleavage reactions. There are many solid-phase sequences with resin attachment via carboxylic acids, followed by elaboration to a free amine five centers away... 

Millipore Australia for support of our solid-phase sequencing program. This work summarises recent work in our group and elements of it (including Fig.l A and C) have been published previously [8]. 

R. Aebersold, G.D. Pipes, R.E. Wettenhall, H. Nika, and L.E. Hood. 1990. Covalent attachment of peptides for high sensitivity solid-phase sequence analysis Ana/. Biochem. 187 56-65. (PubMed)... 

The primary amino acid sequence of polypeptide is routinely determined using the commercially available gas-liquid-phase sequencers [1, 2] and solid-phase sequencers [3, 4] based on the Mman degradation chemistry [.5j. These instruments can routinely obtain the primary amino acid sequence from 10 to 100 pmol of polypeptide. However, the need for higher sequencing sensitivity remains, as Kent et al. [6] have pointed out that rare proteins may only be present at the 30-300 fmol level on 2D-polyacrylamide gels. 

Solid-phase sequencer, an apparatus for peptide/protein sequence analysis. Automated primary structure determination of peptides and proteins using the solid-phase sequencing method is based on cova-... 

In 1973, Wachter and Machleidt (Wachter et a/., 1973 Machleidt et a/., 1973) described the use of aminoglass supports, which for the first time permitted solid-phase sequencing of proteins. The supports are prepared by silylating controlled-pore glass beads (e.g.. Corning CPG-10,200-400 mesh) with 3-aminopropyltrimethoxysilane (Wachter et a/., 1973) or iV-(2-amino-ethyl)-3-aminopropyltriethoxysilane (Bridgen, 1975) (see Fig. 8). The resulting... 

The design and construction of the solid-phase peptide sequencer have been discussed in detail (Laursen et ai, 1975). Basically, the instrument consists of a thermostatted reaction column, which holds the resin-bound peptide, and a series of pumps and valves that deliver reagents and solvents to the column. After each cycle, the liberated thiazolinones are collected in a fraction collector. The instrument is fully automatic and completes a cycle in 60-90 min, depending on how it is programmed. Solid-phase sequencers based on the design of Laursen (1971 Laursen et a/., 1975) are available from Sequemat, Inc. (Watertown, Massachusetts), LKB Instruments (Rockville, Maryland), and Rank-Hilger (United Kingdom). 

Solid-phase sequencing has great potential for microsequencing, especially of hydrophobic polypeptides. Since the peptide is covalently bound, me-... 

In the future, the technique of solid-phase sequencing will be most aided by improved peptide coupling methods and a reduction in the side reactions of the Edman degradation. 

Chapter 6 Solid-Phase Sequencing of Peptides and Proteins... 

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