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
`
`(j)
`
`Edward Arnold
`
`PFIZER EX. 1090
`Page 1
`
`

`

`©Keith Wilson and Kenneth H. Goulding, 1986
`
`First published in Great Britain 1975 by
`Edward Arnold (Publishers) Ltd, 41 Bedford Square, London WC1B 3DQ
`
`Edward Arnold (Australia) Pty Ltd, 80 Waverley Road, Caulfield East,
`Victoria 3145, Australia
`
`Edward Arnold, 3 East Read Street, Baltimore, Maryland 21202, U.S.A.
`Reprinted 1976, 1979
`Second edition 1981
`Reprinted with corrections 1983
`Reprinted 1984
`Third edition 1986
`
`British Library Cataloguing in Publication Data
`
`A Biologist's guide to principles and
`techniques of practical biochemistry.--
`3rd ed.-(Contemporary biology)
`1. Biological chemistry--Technique
`I. Wilson, Keith, 1936-
`II. Goulding,
`III. Series
`Kenneth H.
`574.19'2'028
`QP519.7
`
`ISBN 0-7131-2942-5
`
`All rights reserved. No part of this publication may be reproduced, stored in a
`retrieval system, or transmitted in any form or by any means, electronic,
`photocopying, recording, or otherwise, without the prior permission of
`Edward Arnold (Publishers) Ltd.
`
`Text set in 10/l1pt Times Compugraphic
`by Colset Pte. Ltd., Singapore
`Printed and bound in Great Britain by Richard Clay Ltd., Bungay, Suffolk
`
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`170 Molecular biology techniques
`
`5'--- TACGCTCG- 32P 3'
`
`Single-stranded DNA,
`labelled only at its 3' end
`
`Modification of 'C' using hydrazine;
`this removes base, leaving ribosyl urea
`
`--- T ACGCTCG-32P
`
`--- T ACGCTCG-32P
`
`!
`--- TACGCTCG-32P ! Cl..v•ge" mod ;fled b.,e,, "''"• plpe,ldl"'
`
`G-32p
`
`TCG-32P
`GCTCG-32P
`
`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 modified and
`cleaved at G, G + A, and T + C, and the products would be separated on the sequencing gel
`alongside those from the 'C' reactions.
`
`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(cid:173)
`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 M13 cloning has been set up in a
`laboratory, it provides a very convenient and rapid way to obtain single(cid:173)
`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 171
`
`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 5.9.5).
`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 V8-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 sequences 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 amin6 acid. By repeated cycles of Edman degradation and iden(cid:173)
`tification of product, the polypeptide can be sequenced.
`In the Edman reaction (Fig. 5.15) the polypeptide is treated with phenyl(cid:173)
`isothiocyanate (PITC), which reacts with theN-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 Dansyi-Edman procedure (Fig. 5.16) is highly sensitive,
`allowing as little as 1 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
`
`N-terminus
`
`C-terminus
`H2N- a,- a2- a3--- an- COOH
`
`PITC
`
`polypeptide
`
`Coupling reaction
`
`OHN -a,-a2-a3--- an-COOH
`
`PTC peptide
`
`Cleavage reaction
`
`Thiazolinone
`
`tCoovorn;oo reoot;oo
`
`Repeat for identification
`of next amino acid
`
`PTH amino acid--------J~- 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 about 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(cid:173)
`rupted by introns. Ironically, DNA sequencing, rather than protein sequenc(cid:173)
`ing, has sometimes been used 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
`
`)
`
`(
`
`...___ __ ..__Edman reactions__)
`(removes N-terminal amino acid)
`
`React with dansyl chloride
`
`t
`
`Dansyl- a1 - a2- a3--- an- COOH'
`~ acid hydrolysis
`
`Dansyl-a 1 plus free amino acids
`
`t
`
`Analyse by TLC to identify dansyl amino acid (fluorescent in uv light)
`
`Fig. 5.16 Dansyi-Edman procedure. Only theN-terminal amino acid becomes dansylated,
`and can therefore be identified by TLC. The Edman degradation is used to 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 yeats 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(cid:173)
`cule may be much less than its total length if some sequences are repetitive,
`but complexity will equal total length if all sequences are unique, appearing
`only once in the genome. In practice, the DNA is first cut randomly into
`fragments about 1 kb in length (Section 5.9.2), and is then completely
`denatured by heating above its Tm. Renaturation at a temperature about
`woe 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 A DNA (genome
`size 49 kb) will contain many more copies of the same sequence per unit
`
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