Differential Screening and Differential Display Technique for Isolation of Genes,mRNA,Reverse Transcriptase,Genomic DNA,Hybridized

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Isolation, Sequencing and Synthesis of Genes Isolation, Sequencing and Synthesis of Genes - Introduction Isolation of Genes Isolation of Genes Coding for Specific Proteins Isolation of Genes with Known or Unknown Products having Tissue Specific Expression Isolation of Genes with Known or Unknown Products Using DNA or RNA Probes Use of Heterologus Probes Use of Synthetic Probes Isolation of Genes Coding Use of Transposable Elements Transposon Tagging T-DNA-Insertion Mutagenesis for Isolation of Plant Genes Promoter Enhancer and Gene Trap for Isolation of Genes Differential Screeing and Differential Display Technique for Isolation of Genes Subtractive Hybridizatioon for Gene Isolation Map Based Cloning for Gene Isolation Isolation of Novel Genes by Asis Data at JNU New Delhi

Home >> Biotechnology and Genomics >> Isolation, Sequencing and Synthesis of Genes >> Differential Screening and Differential Display Technique for Isolation of Genes

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Differential Screening and Differential Display Technique for Isolation of Genes
A number of genes with unknown gene products are expressed in specialized tissues. Mutations in these gene may also not result in easily detectable penotypic changes. Isolation of such genes can be achieved through ‘differential screening’. In this technique mRNA is prepared from contrasting tissues of plants such as the following;
(i) tissues/plants exposed to different environmental conditions (e.g., drought environmental conditions (e.g., drought);
(ii) tissues, which differ in function,
(iii) plants at different developmental stages.

In all these cases, two samples of mRNAs derived from two tissues or two plants should differ and the mRNA species which is not common in both the samples will be identified through differential screening using the following steps
(i) mRNA is used for synthesis of cDNA using reverse transcriptase;
(ii) cDNA thus obtained from both samples of mRNA are labelled and used for sequential probing of a genomic DNA library;
(iii) clones that are more strongly hybridized with the cDNA from one mRNA sample than with that from another contain genes that are differentially expressed in the samples.

One of the limitations of differential screening is that the techniques can be used for only those genes that have high expression, and may not prove suitable for those differentially expressed genes that have low expression (low abundance of mRNA). For such genes, another technique called ‘differential display’ is used.

Different steps involved in iolation of a gene using the technique of differential screening

Different Steps Involved in Isolation of a Gene Using the Technique of Differential Screening

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The technique of differential display makes use of polymerase chain reaction (PCR) to amplify rare cDNAs. Following steps are involved isolate polyA mRNA form two sources as above (call them A and B) and run the following reactions separately with each of the two samples; (ii) conduct RT-PCR (reverse transcribed-PCR) using as primers 12 possible oligonucleotides of sequence T(11)XY (XY stand for any two of the four bases, thus giving 12 possible dinucleotides excluding XY will select the mRNAs, thus providing a means of differential amplification; (ii) the RT-PCR product is amplified using a pair of primers, one of them being a random 10-mer and the other being the oligonucleotide used earlier in RT-PCR.

Different Steps involved in isolation of a gene using the technique of differential display

Diffrent Steps Involved in Isolation of a Gene Using the Technique of Differential Display

This will allow amplification of only a fraction of RT-PCR products, including only those to which random 10-mer anneals. (iv) the PCR products obtained are separated on polyacrylamide gel electrophoresis (PAGE); it will be noticed that most of the PCR products are present in equal amounts in both samples but some will differ in abundance and still other will be unique in one sample (being absent in the other sample); (v) the unique bands or the bands showing differential expression are excised and reamplified using the same pair of primers as used in step (iii) above.

The reamplified product is cloned and used as a probe for screening cDNA or genomic DNA libraries. Since several genes may have similar patterns of expression in two tissues or two plants sampled, additional criteria will be needed to confirm the identify of isolated gene; this can be achieved by construction of transgenic plants expressing an antisense gene construct.

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