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A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.

Kuvbachieva AA, Goffinet AM - BMC Mol. Biol. (2002)

Bottom Line: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides.Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products.We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium. kuv1302@yahoo.co.uk

ABSTRACT

Background: cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.

Results: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development.

Conclusion: We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

No MeSH data available.


Agarose gel electrophoresis (1.5% NuSieve, FMC) of representations and a first round RDA product DP-1 obtained using the modified protocol.A. Both representations of normal (lane 1) and scrambler (lane 2) brain transcripts (used respectively as driver and tester) show a smear between 200 and 1100 bp. B. After the first round of subtractive hybridization/amplification, product DP-1 obtained using the proposed modified protocol exhibits reduced complexity with visible bands (lane 2). The negative control of the reaction is loaded on lane 1. C. By comparison, the usual RDA protocol yields a smear in the DP1. D. and discrete bands only in the DP2 product (lane2). On lane 1 is the control reaction. The size o the photos was determined using 100 bp DNA marker (Invitrogen).
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Figure 2: Agarose gel electrophoresis (1.5% NuSieve, FMC) of representations and a first round RDA product DP-1 obtained using the modified protocol.A. Both representations of normal (lane 1) and scrambler (lane 2) brain transcripts (used respectively as driver and tester) show a smear between 200 and 1100 bp. B. After the first round of subtractive hybridization/amplification, product DP-1 obtained using the proposed modified protocol exhibits reduced complexity with visible bands (lane 2). The negative control of the reaction is loaded on lane 1. C. By comparison, the usual RDA protocol yields a smear in the DP1. D. and discrete bands only in the DP2 product (lane2). On lane 1 is the control reaction. The size o the photos was determined using 100 bp DNA marker (Invitrogen).

Mentions: In the modification of cDNA-RDA, schematised in Fig. 1B, the classical protocol is followed to generate tester and driver representations from normal and mutant brain poly(A)+-RNA. Following DpnII cleavage and prior to subtractive hybridization, the 5' protruding ends of the driver are filled-in by normal dNTPs using Klenow. After hybridization, the recessed ends of duplexes, containing tester fragments, are protected from degradation by Exo III by filling with α-thio-dNTPs, and Exo III is used to digest the heteroduplexes and driver homoduplexes that are unprotected by α-thio-dNTPs [4]. Single stranded fragments and single stranded extensions from the ends of duplexes are degraded by MBN. Next, the mixture is subjected to the 28 cycles of amplification in the same PCR conditions as in the classical RDA protocol, to generate a difference product. In our experience, this simple modification, a variant of which was proposed before [5], is able to generate a difference product that appears as discrete bands on agarose gel after the first round of hybridization (Fig. 2).


A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.

Kuvbachieva AA, Goffinet AM - BMC Mol. Biol. (2002)

Agarose gel electrophoresis (1.5% NuSieve, FMC) of representations and a first round RDA product DP-1 obtained using the modified protocol.A. Both representations of normal (lane 1) and scrambler (lane 2) brain transcripts (used respectively as driver and tester) show a smear between 200 and 1100 bp. B. After the first round of subtractive hybridization/amplification, product DP-1 obtained using the proposed modified protocol exhibits reduced complexity with visible bands (lane 2). The negative control of the reaction is loaded on lane 1. C. By comparison, the usual RDA protocol yields a smear in the DP1. D. and discrete bands only in the DP2 product (lane2). On lane 1 is the control reaction. The size o the photos was determined using 100 bp DNA marker (Invitrogen).
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Related In: Results  -  Collection

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Figure 2: Agarose gel electrophoresis (1.5% NuSieve, FMC) of representations and a first round RDA product DP-1 obtained using the modified protocol.A. Both representations of normal (lane 1) and scrambler (lane 2) brain transcripts (used respectively as driver and tester) show a smear between 200 and 1100 bp. B. After the first round of subtractive hybridization/amplification, product DP-1 obtained using the proposed modified protocol exhibits reduced complexity with visible bands (lane 2). The negative control of the reaction is loaded on lane 1. C. By comparison, the usual RDA protocol yields a smear in the DP1. D. and discrete bands only in the DP2 product (lane2). On lane 1 is the control reaction. The size o the photos was determined using 100 bp DNA marker (Invitrogen).
Mentions: In the modification of cDNA-RDA, schematised in Fig. 1B, the classical protocol is followed to generate tester and driver representations from normal and mutant brain poly(A)+-RNA. Following DpnII cleavage and prior to subtractive hybridization, the 5' protruding ends of the driver are filled-in by normal dNTPs using Klenow. After hybridization, the recessed ends of duplexes, containing tester fragments, are protected from degradation by Exo III by filling with α-thio-dNTPs, and Exo III is used to digest the heteroduplexes and driver homoduplexes that are unprotected by α-thio-dNTPs [4]. Single stranded fragments and single stranded extensions from the ends of duplexes are degraded by MBN. Next, the mixture is subjected to the 28 cycles of amplification in the same PCR conditions as in the classical RDA protocol, to generate a difference product. In our experience, this simple modification, a variant of which was proposed before [5], is able to generate a difference product that appears as discrete bands on agarose gel after the first round of hybridization (Fig. 2).

Bottom Line: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides.Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products.We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium. kuv1302@yahoo.co.uk

ABSTRACT

Background: cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.

Results: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development.

Conclusion: We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

No MeSH data available.