Methods for sequence determination

Derivatization of amino acids with phenylisothiocyanate (XXXXII) to give phenylthiohydantoins (XXXXIIl) is utilized in the Edman method for sequence determination of peptides and proteins. The first step, performed under mildly basic conditions, may be regarded in principle as an acylation ... 

Methods for sequence determination by sequential d radation from the C-terminal end have in neral not been well developed. In an attempt to improve this situation. Louden and co-workers have investigated various chemical methods for determining C-terminal residues and degrading peptides from the C-terminal end. - ... 

Giger and Schnaffer [69] described a glass capillary gas chromatographic method for the determination of polyaromatic hydrocarbons in lake and river sediments. Polyaromatic hydrocarbons are isolated by a sequence of solvent... 

Paul Berg, Walter Gilbert, and Frederick Sangerb Chemistry Recombinant DNA, methods of sequence determination for DNA... 

Since the development of the rapid methods for sequencing DNA, many mRNA sequences have been determined by using reverse transcriptase to make a cDNA strand complementary to the RNA. The cDNA is then sequenced.649 Rapid sequencing methods parallel to those used for DNA have also been devised.650-652... 

I n the previous three chapters we described the structures of amino acids and proteins, and in two cases we examined how these structures relate to their function. Some of the methods for structure determination were also discussed (e.g., sequence analysis in chapter 3 and x-ray diffraction in chapter 4). To analyze the structure of a protein we must isolate it from the complex mixture in which it exists in whole cells. The primary object of this chapter is to acquaint you with techniques used for protein purification. Because these procedures are often used for protein characterization as well, they will add to your repertoire of methods for protein characterization. 

Because the (one-dimensional) sequence of a biological macromolecule determines its ultimate 3-D structure, the detection of sequence similarity implies a 3-D resemblance as well as an evolutionary relationship, and it is therefore relevant to review briefly methods for sequence comparison. However, as we shall see below, the converse is not the case and it is possible for proteins to have very similar 3-D structures without there being any sequence similarity. It is the detection of these more distant structural relationships that form our main subject. 

For DNA-based test methods, the extraction procedure cannot be validated independently from the determination system used. There is little or no alternative but to amplify the target DNA within the extracted material in order to determine the quantity and quality of the DNA-sequence actually used for the amplification reaction. Of course, there are several methods suitable for the determination of the total amount of DNA, but in the subsequent amplification reaction only a small fragment of the DNA is amplified the abundance of this small fragment can only be measured after appropriate amplification. Moreover, the total quantity of DNA in some samples is so small that the sensitivity of methods for the determination of total DNA is not sufficient. 

The intent of this limited protocols section is not to provide the reader with an optimal protocol for sequence determination. The protocols listed are presented because they work reasonably well they give the reader a starting point. However, they are not the final word. This is an area of rapid development, as reflected by frequent publication of improved methods. However, there have been few side-by-side comparisons of protocols. Until such comparisons are made, there can be no specific recommendation. 

With the advent of rDNA technology, the primary amino acid sequences of thousands of proteins have become available through cloning and DNA sequencing. However, the experimental methods for stmcture determination described above are labor- and time-intensive, and are also inapplicable to many proteins for technical reasons discussed earlier. Thus, the nmnber of known sequences far exceeds the number of... 

Because several excellent, nonenzymatic methods are available for amino-terminal end group or sequence analysis, aminopeptidase is not the first choice by many workers for sequence determinations. Aminopeptidase is difficult and expensive to prepare and it remains one of the few proteolytic enzymes used in structural studies that has not been crystallized or prepared in homogeneous form. Finally, it should be emphasized that preparations of aminopeptidase which have a low specific activity often contain other proteolytic activities and care should be exercised in their use (Hill and Smith, 1958 Smyth et al., 1962). 

Figure 1.13 shows the spectrum of a hexapeptide [181] as its permethy-lated derivative with the ions indicating the amino acid order. The rapid decrease in the intensity of the important sequence-determining ions at higher mass is evident and a principal reason why MS often requires several times as much material as the common micro wet chemical methods for sequencing small peptides. The chemical ionization mass spectra of some N -acyl permethylated simple peptides show a much more even distribution of the sequencing peaks and hence require a lower sample level than for El spectra [187]. This may well prove of value in the future. Cl has also been applied directly to peptides [188] where up to six amino acids units have been introduced by the direct insertion probe. 

Frederick Sanger (1918- ) was born in Gloucestershire, England, and received his Ph.D. at the University of Cambridge in 1943. He was awarded the Nobel Prize in chemistry in 1958 for his determination of the structure of insulin, and in 1980 he became only the fourth person ever to win a second Nobel Prize. This second prize was awarded for his development of a method for sequencing nucleotides in DNA. 

A modified DEPT experiment, DEPTQ, recently was developed that permits the detection and display of nonprotonated nuclei, such as quaternary carbons, and that possesses the editing features and polarization transfer sensitivity advantages of the DEPT sequence. The DEPTQ experiment promises to become an important method for the determination molecular structures. 

In conclusion immunohistochemistry is a quick method for p53 determination. This seems to be very suitable for detection of the majority of point mutations, whereas deletions and stop codons to a large extent will be missed. The pros and cons have been discussed recently elsewhere and sequencing was still considered to be the gold standard (67). 

There are more than 10,000 known sequences of proteins, but there are barely a thousand whose three dimensional (3D) structures have been determined, mostly by x-ray diffraction. With the development of multidimensional NMR (nuclear magnetic resonance) (Ernst et al., 1987) and the improvement in computing power, as well as the development of suitable computational algorithms, NMR spectroscopy has emerged as a second important method for structure determination of moderately sized proteins (up to M.W. 37,000 iJius far) in solution. 

As of this writing, 50 years have passed since Pehr Edman published his first note on chemical degradation of peptide chains, A Method for the Determination of the Amino Acid Sequence in Peptides. As testament to the continuing relevance of this technique, the following job announcement was recently posted. 

Edman, R, A method for the determination of the amino acid sequence in peptides. Arch. Biochem., 22, 475 76, 1949. 

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