Isolating a gene
Pro Research Analysisby 
Searched over 200M research papers
Techniques for Isolating Genes: A Comprehensive Overview
Antibody Probes for Gene Isolation
Efficient Gene Isolation Using Antibody Probes
A sensitive technique has been developed for cloning genes using antibodies as probes. This method employs the lambda gt11 expression vector, which allows the insertion of foreign DNA into the beta-galactosidase gene, facilitating the synthesis of hybrid proteins. Screening for antigen-producing clones is efficient in Escherichia coli hflA mutant cells, which produce detectable antigen quantities upon induction. This vector also aids in isolating proteins specified by previously cloned genes, with hybrid proteins accumulating in lon mutant strains, making large-scale purification feasible1.
Differential cDNA Library Screening
Isolation of Novel Mammalian Genes
A differential cDNA library screening strategy has proven effective in isolating novel genes from mouse testis. By focusing on cDNA clones containing rare sequences, researchers achieved a higher success rate in identifying new genes compared to random cDNA cloning methods. This approach significantly increases the probability of isolating previously uncharacterized mammalian genes2.
PCR and Near-Isogenic Lines
Marker Identification Linked to Resistance Genes
A method utilizing random primers and near-isogenic lines has been developed to isolate DNA sequences linked to important plant genes. This technique, demonstrated in tomato lines differing by a resistance gene, uses synthetic primers to amplify random genomic sequences. The identified linked sequences are useful for detecting target genes in plant populations and can serve as starting points for chromosome walking to isolate the gene3.
Retroviral Expression Cloning
Isolating Genes Encoding Cell Surface Antigens
Retroviral vectors offer a powerful means of isolating genes encoding cell surface molecules (CSM). A novel method using retroviral cDNA libraries and a selection strategy involving monoclonal antibodies and magnetic beads has successfully isolated several cDNAs encoding CSMs. This approach confirms the utility of retroviral cDNA libraries for expression cloning of genes encoding cell surface antigens4.
Thermostable Restriction Endonucleases and PCR
Efficient Isolation of C. elegans Deletion Strains
A novel modification of PCR-based methods using thermostable restriction enzymes has increased the efficiency of isolating small targeted deletions in Caenorhabditis elegans. This method blocks the synthesis of wild-type PCR products, allowing only deletion products to be amplified. This increased efficiency makes gene knock-out screens more practical for smaller laboratories5.
Transposable Elements for Gene Tagging
Gene Isolation Using High Copy Number Transposable Elements
A method has been developed to isolate genes tagged by high copy number transposable elements, such as dTph1 in Petunia hybrida. This technique involves differential screening of cloned inverse PCR products from mutated plants, yielding probes for the mutated genes. This method is applicable to other transposable elements like Mu and Ds in maize6.
Ds Elements in Maize for Gene Tagging
Protocols for isolating genes in maize using Ds transposons marked with a GFP transgene have been described. The GFP marker facilitates phenotypic scoring, and unique primers on the Ds element aid in isolating adjacent DNA by PCR. This approach allows targeted tagging of most genes from a nearby Ds-launching platform7.
Genomic DNA Libraries
Isolation of Structural Genes from Eukaryotic DNA Libraries
A procedure for isolating eukaryotic structural genes involves constructing and screening cloned libraries of genomic DNA. Large DNA fragments are joined to phage lambda vectors, packaged into viable phage particles, and amplified to establish a permanent library. This method has successfully isolated structural genes from Drosophila, silkmoth, and rabbit genomic DNA8.
Genetic Selection in Yeast
Isolating cDNAs Encoding Secreted Proteins
A rapid technique for isolating cDNAs encoding secreted proteins uses a genetic selection in Saccharomyces cerevisiae. A cDNA cloning vector with a modified invertase gene is used in a yeast strain lacking endogenous invertase. Heterologous secreted genes restore secretion, allowing yeast growth on specific sugars. This method enables the identification of novel secreted proteins from cDNA libraries10.
Conclusion
The isolation of genes is a critical step in genetic research, and various techniques have been developed to enhance efficiency and specificity. From antibody probes and differential cDNA screening to PCR-based methods and retroviral expression cloning, each method offers unique advantages for different research needs. These advancements continue to facilitate the discovery and characterization of novel genes across various organisms.
Sources and full results
Most relevant research papers on this topic
Efficient isolation of genes by using antibody probes.
This method effectively clones genes and isolates unknown proteins encoded by cloned DNA, using antibodies as probes.
Highly CitedIsolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.
Focusing on clones with rare sequences significantly improves the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries.
Highly CitedRapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines.
This method, using random primers and near-isogenic lines, rapidly identifies DNA sequences linked to important plant genes, aiding in plant breeding and genetic studies.
Highly CitedA powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning.
This study presents a novel method for rapidly isolating genes encoding cell surface molecules using retroviral expression cloning, enabling the identification of previously unclustered mAbs.
Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR
This study developed a novel modification of PCR-based methods using thermostable restriction enzymes, enabling efficient isolation of small targeted deletions in C. elegans, making it more practical for small laboratories to undertake gene knock-out screens.
A general method to isolate genes tagged by a high copy number transposable element.
This method effectively isolates mutated genes in Petunia hybrida plants by identifying unique flanking sequences in cloned inverse polymerase chain reaction products.
Gene tagging with engineered Ds elements in maize.
Engineered Ds elements with GFP transgenes can effectively tag genes in maize, enabling targeted tagging of most genes from nearby Ds-launching platforms.
The isolation of structural genes from libraries of eucaryotic DNA
This study presents a method for eucaryotic structural gene isolation using cloned libraries of genomic DNA, revealing the presence of closely linked beta-globin genes in Drosophila, silkmoth, and rabbit species.
In Vitro StudyHighly CitedIsolating RNA is easy and fun.
This study presents a novel method for cloning Neurospora nuclear genes using plasmid pRAL1 and sib selection, offering improved yields and a more efficient method for RNA isolation.
A genetic selection for isolating cDNAs encoding secreted proteins.
This study presents a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins in yeast, enabling the identification of novel secreted proteins without specific bioassays.
Highly Cited