DECHNICAL
M o l e c u l a r biology started a new era in the study o f higher eukaryotic genomes. The amplification or cloning of DNA based on the ability of bacteria to replicate foreign DNA provided sufficient quantities o f otherwise undetectable amounts of genetic material for molecular characterization. By this means, the primary structure of numerous genes has been obtained. However, cloning of DNA in bacteria has the severe d r a w b a c k [hat vital information about the native state of a gene is unavoidably lost. For instance, specific DNA-protein interactions are disrupted as a result of cloning. In addition, the methylation state of cytosine residues at specific locations in the eukaryotic g e n o m e is thought to influence the regulation of gene expression, but this was difficult to investigate since 5-methylcytosine is substituted by cytosine in cloned DNA. The obvious way to overcome these problems is to work directly with genomic DNA. Until recently, methylation of DNA was detected by comparing the DNA digestion pattern obtained with pairs of restriction endonuclease isoschizomers, one of which could not cleave DNA carrying 5-methylcytosine within its recognition sequence. This meant, of course, that the researcher's view of the DNA relied on the biologically significant 5-methylcytosine residue being within the recognition sequence of a methylation-
[gOCUS
Studying DNA modifications and DNA-pr0tein interactions in vivo a window onto the native genome HANS PETER SALUZ~KARIN WIEBAUER AND ANDREW WALLACE The study of native genomes has been greatly facilitated by the use of direct genomic sequencing and footprinting strategies. Tbis review provides an overview of the techniques involved and presents some highlights of the biological results obtained with these methods. sensitive endonticlease. This shortcoming called for the d e v e l o p m e n t of a technique that would allow the detection of all 5-methylcytosines in genomic DNA, a d e m a n d that can ideally be fulfilled by sequencing. This review deals with the d e v e l o p m e n t of appropriate
Hc;n Schematic diagram of the original genomic sequencing procedure. Initially, the DNA is digested to completion with a restriction endonuclease. As the sequence of interest must already be known from cloning experiments. the restriction enzyme is chosen so as to provide fragments of defined length that contain the target sequence. These are subjected to the chemical sequencing reactions, which consist of two steps: a base-specific chemical modification, carried out tinder conditkms in which only a random subset of bases react, followed by a cleavage at all mcx_lifiedsites. The result is a set of subfragments covering the whole sequence base by base. The subfragments of each sequencing reaction are separated by size on a polyacrylamide gel, and subsequently transferred to a nylon membrane, to which they are covalently linked by UV irradiation. The filter-bound subfragments containing the sequence of interest are specifically hybridized with a single-stranded radioactively labeled I)NA probe complementary to one end of the target fragments. Upon processing of the membrane and exposure to an X-ray film, the target sequence can be visualized. The position of 5methylcytosine, which is not modified by the C-specific reagent, is revealed as a gap (indicated by an arrowhead to the right of the X-ray film box) in the C ladder of the genomic sequence (C~,n), compared with that of the cloned DNA (C). The thick lines represent the target sequence, while its probe region is shown as an ()pen box at one end of the target sequence. The tilin lines are the remaining unrecognized 1)NA fragments.
Cells Rei~icliS:iai:ilstI
Chemicalcleavage ÷
Hybridisationof labelled single-strandedprobeto membrane-boundDNA Gel electrophoresis
iiG.iT.c--GG"T'cc c %.nlGG.AT.CC__ m
Sequencing gel
r i g JILY
f991
V(_)L. 7 N(), "7
Membrane
~
i,q
X-ray
film
i[liECHNICAL ri~ o c u s which are now radiolabeled, are separated by size on a standard sequencing gel and directly detected by autoradiography. Analysis of genomic sequences is simplified by this method, because it involves fewer C steps and results are obtained 5' I 3' more rapidly. A promising C 3' One way to increase, the Church and Gilbert 1 deI 5" amount of signal further was serve the credit for ingeniously ! 5-mC 3' to incorporate the polymcombining known techniques I erase chain reaction (PCR) to produce a sequencing proII into genomic sequencing procedure that provides the priC-specific reaction cedures, allowing exponential mary structure of uncloned I, amplification of the chemigenomic DNA of k n o w n secally cleaved DNA. This was quence, including the position not immediately feasible, {}f 5-methylcytosines. Their , however, as the products of strategy, published in 1984, 3' linear amplification start at the was to apply chemical se5'-end with a defined primer, quencing reactions 2 to puri3' but end at wherever chemical fied genomic DNA and to use , 3' cleavage occurred. Thus, amindirect end-labeling to visualplification of each fragment ize the sequence of interest. would have required the In the legend to Fig. 1 we Linear amplification with addition of a separate primer {}utline the points of this prolabelled primer and Taq to each fragment. This recedure that are essential for polymerase quirement was circumvented understanding the rationale by the method of ligationbehind more recent methods. mediated PCR, introduced toAlthough there is no specific wards the end of 1989 ~,9. In -, reaction to detect 5-methylthis method, a short doublecytosine directly - since the stranded DNA oligomer m{}dified base, 5-methylcyto(linker) was ligated to the sine, escapes detection by the primer-extended and thereMaxam and Gilbert sefk}re bhmt-ended chemical quencing reactions - its presr cleavage sites, thus providing ence in genomic I)NA is ina common sequence that ferred by the existence of a Sequencing gel and allowed synthesis in the regap in the genomic seautoradiography verse direction with a single quencing ladder, at a position primer. With a primer site where a band corresponding available at each end of the t{} an unmodified cytosine Reaction scheme of genomic sequencing using linear target sequence, exponential appears in the cloned DNA amplification with a radioactively labeled primer. In amplification then became sequence ladder. The possithe example here, the genomic DNA is subjected to possible. The amplificati(m bility that this gap arises from a C-specific reaction, where only unmethylated products were labeled by a deamination of 5-methylcytosines are chemically m{}dified and subsequently de;Bed, tpon repeated cycles {}f extension with a primer extension s using a cytosine to thymine during radiolabelcd primer and separation {}n a sequencing radioactively marked primer, is{}lation of the genomic I)NA gel, no sequencing fragment c{}rresponding t{~ the separated on a sequencing can be excluded by perposition of the methvlated c}losine is observed. gel and visualized by autoforming a T-specific reaction resulting in a characteristic gap in the sequencing radiography (Fig. 3). This in parallel with tile other ladder. method is s{}mewhat more chemical cleavage reactions. complex in that it involves (For further details, see Ref, 3. more steps and requires tile unbiased efficiency of the This procedure showed such great potential that it was ligase reaction. Nevertheless. genomic sequencing by also applied to plants with large genomes, such as ligati{m-mediated PCR has a great advantage over the maize ~ and parsley ~. other genomic sequencing techniques developed to date, in that it provides greater sensitivity. Recent developments One major disadvantage of all currently available Five years after the method of Church and Gilbert genomic sequencing procedures is the fact that they was described, a new procedure was introduced short sequence ladder, even Primer annealing trol DNA (Fig. -4). with fully efficient amplifiIn the case of / V photocation.) Even if amplification 1 footprinting, the exacl m~,lw'ere used before chemical Primer extension ecular mechanism is m)l cleavage, as in mutatkm understood. In princip!c it r,' studies l°, the prob'lem w o u l d Linker lies on the fact that the extent persist, because the copying ,=¢ ! of photoproduct formation process results in loss of inLinker Iigation u p o n ITV irradiatkm of I)NA formation about the native EB differs between protein-free state of the genomic DNA. ! and protein-hound I)N¢. Thus the desire of many rePrimer annealing and extension Thus, breakage of the I)NA searchers to study the g e n o m e b a c k b o n e
M o l e c u l a r biology started a new era in the study o f higher eukaryotic genomes. The amplification or cloning of DNA based on the ability of bacteria to replicate foreign DNA provided sufficient quantities o f otherwise undetectable amounts of genetic material for molecular characterization. By this means, the primary structure of numerous genes has been obtained. However, cloning of DNA in bacteria has the severe d r a w b a c k [hat vital information about the native state of a gene is unavoidably lost. For instance, specific DNA-protein interactions are disrupted as a result of cloning. In addition, the methylation state of cytosine residues at specific locations in the eukaryotic g e n o m e is thought to influence the regulation of gene expression, but this was difficult to investigate since 5-methylcytosine is substituted by cytosine in cloned DNA. The obvious way to overcome these problems is to work directly with genomic DNA. Until recently, methylation of DNA was detected by comparing the DNA digestion pattern obtained with pairs of restriction endonuclease isoschizomers, one of which could not cleave DNA carrying 5-methylcytosine within its recognition sequence. This meant, of course, that the researcher's view of the DNA relied on the biologically significant 5-methylcytosine residue being within the recognition sequence of a methylation-
[gOCUS
Studying DNA modifications and DNA-pr0tein interactions in vivo a window onto the native genome HANS PETER SALUZ~KARIN WIEBAUER AND ANDREW WALLACE The study of native genomes has been greatly facilitated by the use of direct genomic sequencing and footprinting strategies. Tbis review provides an overview of the techniques involved and presents some highlights of the biological results obtained with these methods. sensitive endonticlease. This shortcoming called for the d e v e l o p m e n t of a technique that would allow the detection of all 5-methylcytosines in genomic DNA, a d e m a n d that can ideally be fulfilled by sequencing. This review deals with the d e v e l o p m e n t of appropriate
Hc;n Schematic diagram of the original genomic sequencing procedure. Initially, the DNA is digested to completion with a restriction endonuclease. As the sequence of interest must already be known from cloning experiments. the restriction enzyme is chosen so as to provide fragments of defined length that contain the target sequence. These are subjected to the chemical sequencing reactions, which consist of two steps: a base-specific chemical modification, carried out tinder conditkms in which only a random subset of bases react, followed by a cleavage at all mcx_lifiedsites. The result is a set of subfragments covering the whole sequence base by base. The subfragments of each sequencing reaction are separated by size on a polyacrylamide gel, and subsequently transferred to a nylon membrane, to which they are covalently linked by UV irradiation. The filter-bound subfragments containing the sequence of interest are specifically hybridized with a single-stranded radioactively labeled I)NA probe complementary to one end of the target fragments. Upon processing of the membrane and exposure to an X-ray film, the target sequence can be visualized. The position of 5methylcytosine, which is not modified by the C-specific reagent, is revealed as a gap (indicated by an arrowhead to the right of the X-ray film box) in the C ladder of the genomic sequence (C~,n), compared with that of the cloned DNA (C). The thick lines represent the target sequence, while its probe region is shown as an ()pen box at one end of the target sequence. The tilin lines are the remaining unrecognized 1)NA fragments.
Cells Rei~icliS:iai:ilstI
Chemicalcleavage ÷
Hybridisationof labelled single-strandedprobeto membrane-boundDNA Gel electrophoresis
iiG.iT.c--GG"T'cc c %.nlGG.AT.CC__ m
Sequencing gel
r i g JILY
f991
V(_)L. 7 N(), "7
Membrane
~
i,q
X-ray
film
i[liECHNICAL ri~ o c u s which are now radiolabeled, are separated by size on a standard sequencing gel and directly detected by autoradiography. Analysis of genomic sequences is simplified by this method, because it involves fewer C steps and results are obtained 5' I 3' more rapidly. A promising C 3' One way to increase, the Church and Gilbert 1 deI 5" amount of signal further was serve the credit for ingeniously ! 5-mC 3' to incorporate the polymcombining known techniques I erase chain reaction (PCR) to produce a sequencing proII into genomic sequencing procedure that provides the priC-specific reaction cedures, allowing exponential mary structure of uncloned I, amplification of the chemigenomic DNA of k n o w n secally cleaved DNA. This was quence, including the position not immediately feasible, {}f 5-methylcytosines. Their , however, as the products of strategy, published in 1984, 3' linear amplification start at the was to apply chemical se5'-end with a defined primer, quencing reactions 2 to puri3' but end at wherever chemical fied genomic DNA and to use , 3' cleavage occurred. Thus, amindirect end-labeling to visualplification of each fragment ize the sequence of interest. would have required the In the legend to Fig. 1 we Linear amplification with addition of a separate primer {}utline the points of this prolabelled primer and Taq to each fragment. This recedure that are essential for polymerase quirement was circumvented understanding the rationale by the method of ligationbehind more recent methods. mediated PCR, introduced toAlthough there is no specific wards the end of 1989 ~,9. In -, reaction to detect 5-methylthis method, a short doublecytosine directly - since the stranded DNA oligomer m{}dified base, 5-methylcyto(linker) was ligated to the sine, escapes detection by the primer-extended and thereMaxam and Gilbert sefk}re bhmt-ended chemical quencing reactions - its presr cleavage sites, thus providing ence in genomic I)NA is ina common sequence that ferred by the existence of a Sequencing gel and allowed synthesis in the regap in the genomic seautoradiography verse direction with a single quencing ladder, at a position primer. With a primer site where a band corresponding available at each end of the t{} an unmodified cytosine Reaction scheme of genomic sequencing using linear target sequence, exponential appears in the cloned DNA amplification with a radioactively labeled primer. In amplification then became sequence ladder. The possithe example here, the genomic DNA is subjected to possible. The amplificati(m bility that this gap arises from a C-specific reaction, where only unmethylated products were labeled by a deamination of 5-methylcytosines are chemically m{}dified and subsequently de;Bed, tpon repeated cycles {}f extension with a primer extension s using a cytosine to thymine during radiolabelcd primer and separation {}n a sequencing radioactively marked primer, is{}lation of the genomic I)NA gel, no sequencing fragment c{}rresponding t{~ the separated on a sequencing can be excluded by perposition of the methvlated c}losine is observed. gel and visualized by autoforming a T-specific reaction resulting in a characteristic gap in the sequencing radiography (Fig. 3). This in parallel with tile other ladder. method is s{}mewhat more chemical cleavage reactions. complex in that it involves (For further details, see Ref, 3. more steps and requires tile unbiased efficiency of the This procedure showed such great potential that it was ligase reaction. Nevertheless. genomic sequencing by also applied to plants with large genomes, such as ligati{m-mediated PCR has a great advantage over the maize ~ and parsley ~. other genomic sequencing techniques developed to date, in that it provides greater sensitivity. Recent developments One major disadvantage of all currently available Five years after the method of Church and Gilbert genomic sequencing procedures is the fact that they was described, a new procedure was introduced short sequence ladder, even Primer annealing trol DNA (Fig. -4). with fully efficient amplifiIn the case of / V photocation.) Even if amplification 1 footprinting, the exacl m~,lw'ere used before chemical Primer extension ecular mechanism is m)l cleavage, as in mutatkm understood. In princip!c it r,' studies l°, the prob'lem w o u l d Linker lies on the fact that the extent persist, because the copying ,=¢ ! of photoproduct formation process results in loss of inLinker Iigation u p o n ITV irradiatkm of I)NA formation about the native EB differs between protein-free state of the genomic DNA. ! and protein-hound I)N¢. Thus the desire of many rePrimer annealing and extension Thus, breakage of the I)NA searchers to study the g e n o m e b a c k b o n e
