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`A Biologist's Guide to
`Principles
`and Techniques of
`Practical
`Biochemistry
`
`Third Edition
`
`Edited by
`Keith Wilson and Kenneth H. Goulding
`
`Contemporary Biology
`
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`BI Exhibit 1090
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`A series of student texts in
`Contemporary Biology
`
`General Editors:
`Professor Arthur J. Willis
`Professor Michael A. Sleigh
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`A Biologist's Guide to
`Principles and Techniques
`of Practical Biochemistry
`
`Third Edition
`
`Edited by
`Keith Wilson
`B.Sc., Ph.D.
`Head of Division of Biological and Environmental Sciences,
`The Hatfield Polytechnic
`
`and
`Kenneth H. Goulding
`M.Sc., Ph.D.
`Head of School of Applied Biology,
`Lancashire Polytechnic
`
`Edward Arnold
`A division of Hodder & Stoughton
`LONDON NEW YORK MELBOURNE AUCKLAND
`
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`© 1986 Keith Wilson and Kenneth H. Goulding
`in Great Britain 1975
`First published
`Reprinted 1976, 1979
`Second edition 1981
`with corrections
`1983
`Reprinted
`Reprinted 1984
`Third edition 1986
`1988
`Reprinted
`in the USA by Routledge, Chapman and Hall, Inc.
`Distributed
`New York. NY IOOOI
`29 West 35th Street,
`
`Library Cataloguing in Publication Data
`British
`
`guide to principles
`and
`A Biologist's
`biochemistry.-
`techniques of practical
`biology)
`3rd ed.-{Contemporary
`chemistry-Technique
`I. Biological
`I. Wilson, Keith, 1936- II. Goulding,
`Kenneth H. Ill. Series
`576.19'2'028 QP519.7
`
`ISBN 0-7131-2942-5
`
`may be reproduced
`No part of this publication
`All rights reserved.
`in any form or by any means, electronically or
`or transmilled
`or any infonnation
`recording
`photocopying,
`including
`mechanically,
`system, without either prior permission
`in
`storage or retrieval
`copying.
`or a licence penniuing restricted
`writing from the publisher
`ln the United Kingdom such licences are issued by the Copyright
`Agency: 33-34 Alfred Pince, London WCIE 7DP.
`Licensing
`
`by Colset Private Ltd,
`Typeset in 10/1 lpt Times Compugraphic
`Printed and bound in Great Britain for Edward Arnold,
`Singapore.
`division
`of
`academic and medical publishing
`the educational,
`Hodder and Stoughton Limited, 41 Bedford Square, London WClB
`3DQ by Richard Clay Ltd., Bungay, Suffolk.
`
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`170 Molecular biology techniques
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`s' ---TACGCTCG -31P 3'
`
`Single-stranded DNA.
`labelled only at its 3' end
`
`Modification of 'C' using hydrazine;
`this removes base, leaving ribosyl urea
`
`t
`i Clo••'9• " modlfi•d b""· ,.;,, p;po.;o;oo
`•• ,, 000· .. ·;·r··
`
`---TACGCTCG-32P
`
`---T ACGCTCG-32P
`
`---TACGCTCG-32P
`
`G-32p
`TCG-32P
`GCTCG-32P
`· .. ·m'""
`
`Separate on sequencing gel alongside products of other
`modification I cleavage reactions (as in Fig. 5. 13)
`
`Fig. 5.14 Maxam and Gilbert sequencing of DNA Only modification and cleavage of
`deoxycytidine is shown, but three more aliquots of the end-labelled DNA would be modifie d and
`cleaved at G. G + A, and T + C, and the products would be separated on the sequencing gel
`alongside tnose from the ·c· ro:dctrons.
`
`to that produced by the Sanger method, since each sample now contains
`radioactive molecules of various lengths, all with one end in common (the
`labelled end), and with the other end cut at the same type of base. Analysis of
`the reaction products by electrophoresis is as described for the Sanger
`method.
`Because the Sanger method produces oligonucleotides which are radio
`actively labelled throughout their lengths, rather than only at one end, the
`molecules can be made a lot more radioactive, and therefore easier to detect;
`so less DNA is needed for sequencing. Once M 13 cloning has been set up in a
`laboratory, it provides a very convenient and rapid way to obtain single
`stranded DNA. For these reasons, dideoxy sequencing of M13-cloned DNA
`is probably the most commonly used sequencing method, though the
`chemical procedure is still used by many laboratories.
`
`5.5.3 Protein sequencing
`
`Although protein sequencing may seem out of place in a section dealing with
`the analysis of DNA, the molecular biologist can often make use of a
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`Analysis of DNA
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`171
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`knowledge of protein sequences when manipulating DNA. If the sequence of
`a protein is known, a gene coding for it can be synthesised chemically (though
`this is usually only worth doing for small polypeptides), or an oligonucleotide
`probe can be synthesised for use in recovering the gene for that protein from a
`gene library (Section S.9.5).
`Since it is currently impossible to sequence a polypeptide longer than about
`100 amino acids, pure proteins must be fragmented to give polypeptides of a
`length which can be sequenced, and these polypeptides must be separated
`from each other prior to sequencing. Fairly specific and limited cleavage can
`be obtained by chemical means. For example, cyanogen bromide cleaves only
`at (rare) methionine residues, BNPS-skatole cleaves at tryptophan, and
`hydroxylamine breaks the linkage between asparagine and glycine. Similarly,
`several proteolytic enzymes, such as trypsin and VS-Protease, have a fairly
`specific site of action, and will therefore generate relatively few cleavage
`products.
`The polypeptides so produced are separated from each other prior to
`sequencing, using such techniques as exclusion chromatography (Section 6.6)
`or HPLC (Section 6.8). Relative positions of the polypeptides within a
`protein can be found by looking for overlaps in the sequence.c; of polypeptides
`generated by different means, and in this way the entire protein sequence may
`be deduced.
`All protein sequencing methods are based on the Edman degradation of
`polypeptides, in which the N-terminal amino acid is specifically removed,
`leaving a polypeptide one amino acid residue shorter. Variations arise in
`the method of identifying the removed amino acid or the newly exposed
`N-terminal amino acid. By repeated cycles of Edman degradation and iden
`tification of product, the polypeptide can be sequenced.
`In the Edman reaction (Fig. 5. l S) the polypeptide is treated with phenyl
`isothiocyanate (PITC), which reacts with the N-terminal amino acid to form
`a phenylthiocarbamyl (PTC) derivative of the polypeptide. Anhydrous
`trifluoroacetic acid is then used to cleave the molecule, giving the
`2-anilino-5-thiazolinone derivative of the N-terminal amino acid and also
`the polypeptide shortened by one residue. The thiazolinone derivative is
`separated from the polypeptide and converted into the more stable
`3-phenyl-2-thiohydantoin (PTH) derivative, which is then identified by
`HPLC or TLC. By repeating this cycle the polypeptide can be sequenced
`from its N-terminal end. The process has been automated, either by
`immobilising the protein on an inert, solid .support (solid-phase sequencers),
`or by keeping the protein spread out in a thin film for maximum exposure to
`reagents (spinning cup sequencers). Such instruments can, under ideal
`conditions, sequence up to 100 residues of a protein.
`The alternative Dansyl-Edman procedure (Fig. 5.16) is highly sensitive,
`allowing as little as I nmole of polypeptide to be sequenced, and it is therefore
`well suited to manual determination of sequences. It uses cycles of the Edman
`reaction to remove N-terminal amino acids sequentially, but, instead of
`identifying the released PTH derivatives, it identifies the newly exposed
`N-terminal amino adds. This is achieved by adding a dansyl group to the
`N-terminal of a very small sample of the polypeptide after each cycle of the
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`172 Molecular biology techniques
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`C-terminus
`H2N-a,-a2-a3- - -an-COOH
`
`N-terminus
`
`0
`PITC \ I polypeptide
`�Coupling reaction
`
`Thiazolinone
`
`ic,,,.�;oo •mdoo
`
`Repeat for identification
`of next amino acid
`
`PTH amino acid----- Identify by HPLC or TLC
`
`Fig. 5.15 Edman reactions. PITC phenylisothiocyanate; PTC, phenylthiocarbamyl; PTH
`3-phenyl-2-thiohydantoin. Note that each cycle of reactions removes one amino acid from the
`N-terminus of the polypeptide.
`
`Edman reaction, followed by cleavage with hydrochloric acid to release a
`dansyl amino acid plus free amino acids. The dansyl derivative can be
`identified by two-dimensional TLC on polyamide plates (Section 6.1.3). Up
`to ab6ut 15 amino acids can be sequenced before the cumulative effects of
`incomplete reactions and side reactions make impossible the unambiguous
`identification of the dansyl amino acid.
`Given the nucleotide sequence of a gene, and our knowledge of the genetic
`code, it is easy to read off the amino acid sequence for which the gene codes,
`provided the correct reading frame is used, and the sequence is not inter
`rupted by introns. Ironically, DNA sequencing, rather than protein sequenc
`ing, has sometimes been us�d to obtain amino acid sequences of proteins,
`especially when the pure protein has not been obtainable in sufficient
`quantities for direct sequencing. However, it should be remembered that a lot
`of effort is involved in the isolation of a specific gene, and this may more than
`offset the rapidity of DNA sequencing. The pace of sequencing is such that
`computers are now used by some laboratories for the analysis of sequencing
`gels, and sequence data banks have been set up to cope with the massive flow
`of information. In spite of this, it will be some time before the human genome
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`Analysis of DNA 173
`
`Repeat to identify next amino acid
`
`(
`
`l
`Edman reactions _)
`
`(removes N-terminal amino acid)
`
`React with dansyl chloride
`
`Dansyl -a, -82-83 • - · 8n-COOH
`
`Dansyl-a, plus free amino acids
`
`� acid hydrolysis
`i
`
`Analyse by TLC to identify dansyl amino acid (fluorescent in uv light)
`
`Fig. 5.16 Dansyl-Edman procedure. Only the N-terminal amino acid becomes dansylated,
`and can therefore be identified by TLC. The Edman degradation is used 10 remove N-terminal
`amino acids one-by-one from the polypeptide, and dansylation allows the identification of each
`newly exposed N-terminal residue.
`
`is completely sequenced. Even at a rate of one base per second, the 3 x 106 kb
`of the haploid genome would take more than 100 years to be sequenced.
`
`5. 5.4 Renaturation kinetics
`
`When preparations of double-stranded DNA are denatured by heat or alkali,
`and then allowed to renature, measurement of the rate of renaturation can
`give valuable information about the complexity of the DNA, i.e. how much
`information it contains (measured in base-pairs). The complexity of a mole
`cule may be much less than its total length if some sequences are repetitive,
`but complexity will equal total length if aJI sequences are unique, appearing
`only once in the genome. In practice, the DNA is first cut randomly into
`fragments about I kb in length (Section 5.9.2), and is then completely
`denatured by heating above its Tm. Renaturation at a temperature about
`10°C below the Tm is monitored either by decrease in absorbance at 260 nm
`(the hypochromic effect), or by passing samples at intervals through a
`column of hydroxyapatite, which will adsorb only double-stranded DNA,
`and measuring how much of the sample is bound. The degree of renaturation
`after a given time will depend on C0, the concentration (in nucleotides per
`unit volume) of double-stranded DNA prior to denaturation, and t, the
`duration of the renaturation.
`For a given C0, it should be evident that a preparation of>. DNA (genome
`size 49 kb) will contain many more copies of the same sequence per unit
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