Hong-Khanh Nguyen - Academia.edu (original) (raw)

Papers by Hong-Khanh Nguyen

Nucleic Acids Research, 1998

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexe... more Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G 4Et C base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51_C). The 16_C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G 4Et C base pairs is mainly due to the sequence effect and not to their AT/G 4Et C ratio content. Thus N-4-ethyl-2′-deoxycytidine (d 4Et C) hybridizes specifically with natural deoxyguanosine leading to a G 4Et C base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d 4Et C can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d 4Et CTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

La presente invention a pour objet une methode de detection de la transformation d'un substra... more La presente invention a pour objet une methode de detection de la transformation d'un substrat (S) en un produit (P), caracterisee en ce que l'on mesure un changement de pM resultant de la difference de pouvoir chelatant du substrat (S) et du produit (P) vis-a-vis d'un ion metallique. Avantageusement, la detection est realisee en mesurant le changement des proprietes spectrales d'un detecteur chimique (L), chelateur d'un ion metallique, resultant d'echanges d'ions metalliques entre le produit (P) et le detecteur chimique (L), lorsque le produit (P) presente un pouvoir chelatant superieur a celui du substrat (S), ou entre le substrat (S) et le detecteur chimique (L), lorsque le substrat (S) presente un pouvoir chelatant superieur e celui du produit (P).

Microbial Biotechnology, 2010

The biodiversity found in the marine environment is remarkable and yet largely unknown compared w... more The biodiversity found in the marine environment is remarkable and yet largely unknown compared with the terrestrial one. The associated genetic resource, also wide and unrevealed, has raised a strong interest from the scientific and industrial community. However, despite this growing interest, the discovery of new compounds extracted from marine organisms, more precisely from microorganisms, is ruled by a complex legislation. The access and transfer of genetic resource are ruled by the Convention on Biological Diversity. One of the three core objectives of this convention is to ensure the fair and equitable sharing of benefits generated by the use of genetic resources and to split these benefits between the different stakeholders. From the discovery of a microorganism to the commercialization of a product, three main stakeholders are involved: providers of microorganisms, e.g. academic institutes, the scientists who will perform R&D on biodiversity, and the industrial companies which will commercialize the final product arising from the R&D results. This article describes how difficult and complex it might be to ensure a fair distribution of benefits of this research between the parties.

The biodiversity found in the marine environment is remarkable and yet largely unknown compared w... more The biodiversity found in the marine environment is remarkable and yet largely unknown compared with the terrestrial one. The associated genetic resource, also wide and unrevealed, has raised a strong interest from the scientific and industrial community. However, despite this growing interest, the discovery of new compounds extracted from marine organisms, more precisely from microorganisms, is ruled by a complex legislation. The access and transfer of genetic resource are ruled by the Convention on Bio-logical Diversity. One of the three core objectives of this convention is to ensure the fair and equitable sharing of benefits generated by the use of genetic resources and to split these benefits between the different stakeholders. From the discovery of a micro-organism to the commercialization of a product, three main stakeholders are involved: providers of micro-organisms, e.g. academic institutes, the scientists who will perform R&D on biodiversity, and the indus-trial companies...

The invention concerns a method for detecting the transformation of a substrate (S) into a produc... more The invention concerns a method for detecting the transformation of a substrate (S) into a product (P), characterised in that it consists in measuring the pM modification resulting from the difference of the chelating power of the substrate (S) and of the product (P) relative to a metal ion. Advantageously, the detection is carried out by measuring the modification of the spectral properties of a chemical detector (L), chelating agent for a metal ion, resulting from the metal ion exchanges between the product (P) and the chemical detector (L), when the product (P) has a chelating power higher than that of the substrate (S), or between the substrate (S) and the chemical detector (L), when the substrate (S) has a chelating power higher than that of the product (P).

The invention discloses a method for producing a hybridisation complex whose stability is substan... more The invention discloses a method for producing a hybridisation complex whose stability is substantially independent of the base composition of two hybridised nucleic acid molecules, consisting in contacting a first nucleic acid molecule with a second nucleic acid molecule under conditions enabling the formation of a specific hybridisation complex. The invention is characterised in that at least one of the four types of bases contained in the sequence of the first and/or of the second nucleic acid molecule is a modified base having pairing properties close to those of the natural base it replaces, such that at least one of the two types of the base pair contained in the composition of the complex comprises a modified base and in that the totality of the base pairs contained in the composition of the said complex has a like stability.

This study was carried out on 4 batch reactors to determine the specific ammonium oxidizing rate ... more This study was carried out on 4 batch reactors to determine the specific ammonium oxidizing rate (SAOR), specific nitrate forming rate (SNFR) and inhibitory degree of nitrifying activities with saline concentrations. Under salt free condition ammonia was consumed during the reaction period within 200 min. When the salt level increased to 10, 20 and 30 g NaClL -1 in reactor, ammonia depletion took 250, 300 and above 350 min, respectively. During concentration above 10 g NaClL -1 , there was nitrite accumulation. Also, at 30 g NaClL -1 ammonia did not depleted and NO2 - -N accumulated until the final reaction. Nitrate formation rates decreased with increasing salt concentration. SAOR and SNFR showed a decreasing trend as salinity concentrations were increased. The SAOR was reduced from 0.2 to 0.08 mg NH4 + N g -1 VSS day -1 as the salt concentration increased from 0 to 30 g NaCLL -1 . Similarly, the SNFR decreased from 0.26 kg NO3 - -N kg -1 VSS day -1 at saline free to 0.1 kg NO3 - -...

Environmental technology, Jan 21, 2017

Isolation of extracellular polymeric substances (EPSs) producing bacterial strains capable of usi... more Isolation of extracellular polymeric substances (EPSs) producing bacterial strains capable of using sludge as low-cost growth substrate was carried out in this study. A total of 110 EPS-producing strains were isolated from different sources, which include sludge of beer and winery wastewater treatment plant (WWTP); young, 2-month-old and 10-year-old leachate. Thirty-seven isolated strains showed good growth in sludge medium with cell count varying from 10(6) to 10(10) most probable number (MPN)/mL and total EPS concentration from 2737 to 6639 mg/L. Twenty-one strains produced EPS with high flocculation activity (FAmax varied from 72.0% to 80.2%). The highest FAmax (80.2%) was observed with EPS produced by strain BES 19, which was isolated from sludge of beer WWTP. Sludge of beer WWTP, young leachate and 10-year-old leachate were good sources for isolation of EPS-producing bacteria.

L Actualite Chimique, 2002

Biodiversity screening and directed evolution are two fruitful complementary approaches for the d... more Biodiversity screening and directed evolution are two fruitful complementary approaches for the discovery and design of novel biocatalysts. Phenomics is a functional HTS technology designed and patented by Proteus for the screening of natural biodiversity as well as biodiversity generated by combinatorial biology. Phenomics is a function to gene structure approach which provides an alternative to genomics and proteomics. To generate novel variants of natural enzymes, a new technology for directed evolution, L-Shuffling, has been designed and patented by Proteus. The thousands of new recombinants generated by L-Shuffling can be further screened for their biochemical characteristics using Phenomics. The whole process of novel biocatalysts discovery and optimization has been automated using commercially available high throughput robotics.

Organic Process Research & Development, 2002

Thermostable esterases are used in biocatalysis for the synthesis of a variety of products, for e... more Thermostable esterases are used in biocatalysis for the synthesis of a variety of products, for example, flavour esters, emulsifiers, or pharmaceutical intermediates. Industrial enzymatic conversions to obtain chemo-, regio-, and stereoselective transformations may require thermostable enzymes active in organic solvents at high temperatures. To find novel enzymes for industrial bioconversions, we have used a new generation of thermostable reporter substrates (CLIPS-O for CataLyst Identification ProcesS per Oxidization) to screen for esterase activity in thermophilic microorganisms belonging to various thermophilic genus. One of the strains (belonging to Thermus genus) exhibiting activity on short-and long-chain ester CLIPS-O substrates has been selected. The genomic library of this strain was screened using a high-throughput functional technology. Positive clones expressing esterases active at high temperature were immediately characterized during screening for chainlength specificity.

Water Science and Technology, 2009

There is a global need to develop low-cost technologies to remove arsenic from water for individu... more There is a global need to develop low-cost technologies to remove arsenic from water for individual household water supply. In this study, a purified and enriched waste material (treated magnetite waste, TMW) from the Trai Cau's iron ore mine in the Thai Nguyen Province in Vietnam was examined for its capacity to remove arsenic. The treatment system was packed with TMW that consisted of 75% of ferrous-ferric oxide (Fe3O4) and had a large surface area of 89.7 m2/g. The experiments were conducted at a filtration rate of 0.05 m/h to treat groundwater with an arsenic concentration of 380 μg/L and iron, manganese and phosphate concentrations of 2.07 mg/L, 0.093 mg/L and 1.6 mg/L respectively. The batch experimental results show that this new material was able to absorb up to 0.74 mg arsenic/g. The results also indicated that the treatment system removed more than 90% arsenic giving an effluent with an arsenic concentration of less than 30 μg/L while achieving a removal efficiency of ...

Tetrahedron Letters, 1997

... RI RI X OHN Rz N OHN Rz D NH NR,NNH R' NNNNI NHp RORH dI d*C d*G d*C d*C RI R2 VC Rl R2 ... more ... RI RI X OHN Rz N OHN Rz D NH NR,NNH R' NNNNI NHp RORH dI d*C d*G d*C d*C RI R2 VC Rl R2 d ... The latter two parameters seem to be important when comparing the stabilities of duplex 1 (Tint = 34C) and 15 (Tnl,s = 29C) having AT and IC base pairs, respectively, which ...

Tetrahedron Letters, 1997

ModifiedG*C,or A*Ubase pairs havebeen incorporatedat the 4ti positionof a duplexcomposedwithnine ... more ModifiedG*C,or A*Ubase pairs havebeen incorporatedat the 4ti positionof a duplexcomposedwithnine basepairs.llre irrfluenceof these modificationson duplexstabilitieshas beenstudiedby absorptionspectrosco #. It has been formal that G4M'C has thermalstabilitysimilarto that of the AS*U basepair. ThusduplexesinvolvingG 'C or As-base pairs exhibitthe sameTm. @ 1997Elsevier Science Ltd.

Nucleic Acids Research, 1999

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA dupl... more The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA + along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilized temperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

Nucleic Acids Research, 1997

The possibility of equalizing DNA duplex stability is essential for the application of sequencing... more The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N-4-ethyl-2′-deoxycytidine (d 4Et C), which hybridizes specifically with natural dG to give a G 4Et C base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G 4Et C base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

Nucleic Acids Research, 2000

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complement... more Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N4-ethyldeoxycytidine (d 4Et C), which hybridises specifically with natural dG to give a G: 4Et C base pair with reduced stability compared to the natural G:C base pair. Substitution by d 4Et C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

Nature Genetics, 1999

Most public-founded large-scale sequencing projects that use cDNAs are primarily using cDNA libra... more Most public-founded large-scale sequencing projects that use cDNAs are primarily using cDNA libraries which are not enriched for full-length cDNAs. Consequently, only a fraction of the resulting ESTs matches the 5´ end of the original transcript. The target of the Genome Science Laboratory of RIKEN is to clone and sequence the largest number possible of full-length mouse cDNAs in two

Nucleic Acids Research, 1998

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexe... more Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G 4Et C base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51_C). The 16_C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G 4Et C base pairs is mainly due to the sequence effect and not to their AT/G 4Et C ratio content. Thus N-4-ethyl-2′-deoxycytidine (d 4Et C) hybridizes specifically with natural deoxyguanosine leading to a G 4Et C base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d 4Et C can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d 4Et CTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

La presente invention a pour objet une methode de detection de la transformation d'un substra... more La presente invention a pour objet une methode de detection de la transformation d'un substrat (S) en un produit (P), caracterisee en ce que l'on mesure un changement de pM resultant de la difference de pouvoir chelatant du substrat (S) et du produit (P) vis-a-vis d'un ion metallique. Avantageusement, la detection est realisee en mesurant le changement des proprietes spectrales d'un detecteur chimique (L), chelateur d'un ion metallique, resultant d'echanges d'ions metalliques entre le produit (P) et le detecteur chimique (L), lorsque le produit (P) presente un pouvoir chelatant superieur a celui du substrat (S), ou entre le substrat (S) et le detecteur chimique (L), lorsque le substrat (S) presente un pouvoir chelatant superieur e celui du produit (P).

Microbial Biotechnology, 2010

The biodiversity found in the marine environment is remarkable and yet largely unknown compared w... more The biodiversity found in the marine environment is remarkable and yet largely unknown compared with the terrestrial one. The associated genetic resource, also wide and unrevealed, has raised a strong interest from the scientific and industrial community. However, despite this growing interest, the discovery of new compounds extracted from marine organisms, more precisely from microorganisms, is ruled by a complex legislation. The access and transfer of genetic resource are ruled by the Convention on Biological Diversity. One of the three core objectives of this convention is to ensure the fair and equitable sharing of benefits generated by the use of genetic resources and to split these benefits between the different stakeholders. From the discovery of a microorganism to the commercialization of a product, three main stakeholders are involved: providers of microorganisms, e.g. academic institutes, the scientists who will perform R&D on biodiversity, and the industrial companies which will commercialize the final product arising from the R&D results. This article describes how difficult and complex it might be to ensure a fair distribution of benefits of this research between the parties.

The biodiversity found in the marine environment is remarkable and yet largely unknown compared w... more The biodiversity found in the marine environment is remarkable and yet largely unknown compared with the terrestrial one. The associated genetic resource, also wide and unrevealed, has raised a strong interest from the scientific and industrial community. However, despite this growing interest, the discovery of new compounds extracted from marine organisms, more precisely from microorganisms, is ruled by a complex legislation. The access and transfer of genetic resource are ruled by the Convention on Bio-logical Diversity. One of the three core objectives of this convention is to ensure the fair and equitable sharing of benefits generated by the use of genetic resources and to split these benefits between the different stakeholders. From the discovery of a micro-organism to the commercialization of a product, three main stakeholders are involved: providers of micro-organisms, e.g. academic institutes, the scientists who will perform R&D on biodiversity, and the indus-trial companies...

The invention concerns a method for detecting the transformation of a substrate (S) into a produc... more The invention concerns a method for detecting the transformation of a substrate (S) into a product (P), characterised in that it consists in measuring the pM modification resulting from the difference of the chelating power of the substrate (S) and of the product (P) relative to a metal ion. Advantageously, the detection is carried out by measuring the modification of the spectral properties of a chemical detector (L), chelating agent for a metal ion, resulting from the metal ion exchanges between the product (P) and the chemical detector (L), when the product (P) has a chelating power higher than that of the substrate (S), or between the substrate (S) and the chemical detector (L), when the substrate (S) has a chelating power higher than that of the product (P).

The invention discloses a method for producing a hybridisation complex whose stability is substan... more The invention discloses a method for producing a hybridisation complex whose stability is substantially independent of the base composition of two hybridised nucleic acid molecules, consisting in contacting a first nucleic acid molecule with a second nucleic acid molecule under conditions enabling the formation of a specific hybridisation complex. The invention is characterised in that at least one of the four types of bases contained in the sequence of the first and/or of the second nucleic acid molecule is a modified base having pairing properties close to those of the natural base it replaces, such that at least one of the two types of the base pair contained in the composition of the complex comprises a modified base and in that the totality of the base pairs contained in the composition of the said complex has a like stability.

This study was carried out on 4 batch reactors to determine the specific ammonium oxidizing rate ... more This study was carried out on 4 batch reactors to determine the specific ammonium oxidizing rate (SAOR), specific nitrate forming rate (SNFR) and inhibitory degree of nitrifying activities with saline concentrations. Under salt free condition ammonia was consumed during the reaction period within 200 min. When the salt level increased to 10, 20 and 30 g NaClL -1 in reactor, ammonia depletion took 250, 300 and above 350 min, respectively. During concentration above 10 g NaClL -1 , there was nitrite accumulation. Also, at 30 g NaClL -1 ammonia did not depleted and NO2 - -N accumulated until the final reaction. Nitrate formation rates decreased with increasing salt concentration. SAOR and SNFR showed a decreasing trend as salinity concentrations were increased. The SAOR was reduced from 0.2 to 0.08 mg NH4 + N g -1 VSS day -1 as the salt concentration increased from 0 to 30 g NaCLL -1 . Similarly, the SNFR decreased from 0.26 kg NO3 - -N kg -1 VSS day -1 at saline free to 0.1 kg NO3 - -...

Environmental technology, Jan 21, 2017

Isolation of extracellular polymeric substances (EPSs) producing bacterial strains capable of usi... more Isolation of extracellular polymeric substances (EPSs) producing bacterial strains capable of using sludge as low-cost growth substrate was carried out in this study. A total of 110 EPS-producing strains were isolated from different sources, which include sludge of beer and winery wastewater treatment plant (WWTP); young, 2-month-old and 10-year-old leachate. Thirty-seven isolated strains showed good growth in sludge medium with cell count varying from 10(6) to 10(10) most probable number (MPN)/mL and total EPS concentration from 2737 to 6639 mg/L. Twenty-one strains produced EPS with high flocculation activity (FAmax varied from 72.0% to 80.2%). The highest FAmax (80.2%) was observed with EPS produced by strain BES 19, which was isolated from sludge of beer WWTP. Sludge of beer WWTP, young leachate and 10-year-old leachate were good sources for isolation of EPS-producing bacteria.

L Actualite Chimique, 2002

Biodiversity screening and directed evolution are two fruitful complementary approaches for the d... more Biodiversity screening and directed evolution are two fruitful complementary approaches for the discovery and design of novel biocatalysts. Phenomics is a functional HTS technology designed and patented by Proteus for the screening of natural biodiversity as well as biodiversity generated by combinatorial biology. Phenomics is a function to gene structure approach which provides an alternative to genomics and proteomics. To generate novel variants of natural enzymes, a new technology for directed evolution, L-Shuffling, has been designed and patented by Proteus. The thousands of new recombinants generated by L-Shuffling can be further screened for their biochemical characteristics using Phenomics. The whole process of novel biocatalysts discovery and optimization has been automated using commercially available high throughput robotics.

Organic Process Research & Development, 2002

Thermostable esterases are used in biocatalysis for the synthesis of a variety of products, for e... more Thermostable esterases are used in biocatalysis for the synthesis of a variety of products, for example, flavour esters, emulsifiers, or pharmaceutical intermediates. Industrial enzymatic conversions to obtain chemo-, regio-, and stereoselective transformations may require thermostable enzymes active in organic solvents at high temperatures. To find novel enzymes for industrial bioconversions, we have used a new generation of thermostable reporter substrates (CLIPS-O for CataLyst Identification ProcesS per Oxidization) to screen for esterase activity in thermophilic microorganisms belonging to various thermophilic genus. One of the strains (belonging to Thermus genus) exhibiting activity on short-and long-chain ester CLIPS-O substrates has been selected. The genomic library of this strain was screened using a high-throughput functional technology. Positive clones expressing esterases active at high temperature were immediately characterized during screening for chainlength specificity.

Water Science and Technology, 2009

There is a global need to develop low-cost technologies to remove arsenic from water for individu... more There is a global need to develop low-cost technologies to remove arsenic from water for individual household water supply. In this study, a purified and enriched waste material (treated magnetite waste, TMW) from the Trai Cau's iron ore mine in the Thai Nguyen Province in Vietnam was examined for its capacity to remove arsenic. The treatment system was packed with TMW that consisted of 75% of ferrous-ferric oxide (Fe3O4) and had a large surface area of 89.7 m2/g. The experiments were conducted at a filtration rate of 0.05 m/h to treat groundwater with an arsenic concentration of 380 μg/L and iron, manganese and phosphate concentrations of 2.07 mg/L, 0.093 mg/L and 1.6 mg/L respectively. The batch experimental results show that this new material was able to absorb up to 0.74 mg arsenic/g. The results also indicated that the treatment system removed more than 90% arsenic giving an effluent with an arsenic concentration of less than 30 μg/L while achieving a removal efficiency of ...

Tetrahedron Letters, 1997

... RI RI X OHN Rz N OHN Rz D NH NR,NNH R' NNNNI NHp RORH dI d*C d*G d*C d*C RI R2 VC Rl R2 ... more ... RI RI X OHN Rz N OHN Rz D NH NR,NNH R' NNNNI NHp RORH dI d*C d*G d*C d*C RI R2 VC Rl R2 d ... The latter two parameters seem to be important when comparing the stabilities of duplex 1 (Tint = 34C) and 15 (Tnl,s = 29C) having AT and IC base pairs, respectively, which ...

Tetrahedron Letters, 1997

ModifiedG*C,or A*Ubase pairs havebeen incorporatedat the 4ti positionof a duplexcomposedwithnine ... more ModifiedG*C,or A*Ubase pairs havebeen incorporatedat the 4ti positionof a duplexcomposedwithnine basepairs.llre irrfluenceof these modificationson duplexstabilitieshas beenstudiedby absorptionspectrosco #. It has been formal that G4M'C has thermalstabilitysimilarto that of the AS*U basepair. ThusduplexesinvolvingG 'C or As-base pairs exhibitthe sameTm. @ 1997Elsevier Science Ltd.

Nucleic Acids Research, 1999

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA dupl... more The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA + along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilized temperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

Nucleic Acids Research, 1997

The possibility of equalizing DNA duplex stability is essential for the application of sequencing... more The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N-4-ethyl-2′-deoxycytidine (d 4Et C), which hybridizes specifically with natural dG to give a G 4Et C base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G 4Et C base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

Nucleic Acids Research, 2000

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complement... more Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N4-ethyldeoxycytidine (d 4Et C), which hybridises specifically with natural dG to give a G: 4Et C base pair with reduced stability compared to the natural G:C base pair. Substitution by d 4Et C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

Nature Genetics, 1999

Most public-founded large-scale sequencing projects that use cDNAs are primarily using cDNA libra... more Most public-founded large-scale sequencing projects that use cDNAs are primarily using cDNA libraries which are not enriched for full-length cDNAs. Consequently, only a fraction of the resulting ESTs matches the 5´ end of the original transcript. The target of the Genome Science Laboratory of RIKEN is to clone and sequence the largest number possible of full-length mouse cDNAs in two