The entire assay relies on the ability of one part of each of the hybrid protein to form a specific interaction that is reasonably stable under physiological conditions with the other. A number of variations of this approach have been developed, but they all rely on the same feature. Screening by databases. The rapid accumulation of sequence information and genetic data often allows scientists to bypass the steps required to isolate cDNAs. For example, if partial protein sequence or partial cDNA sequence is available, searching data bases may result in identifying candidate clones that can be ordered and tested to determine if they are the 'right' clone.
Each of these methods of screening a cDNA library provides a specific screen or assay for cDNAs that may be of interest. Just as it is true that when purifying a protein, one is likely to get what one assays for, in screening a cDNA library one is likely to get what one screens for.
Determining whether the cDNA that has been isolated is indeed the one that is of most interest to the experimentalist requires additional tests. In the absence of understanding of what those tests should be, it will makes little sense to do initial screenings. Likewise, careful consideration of what are the best screens for a specific purpose is likely to result in a more fruitful search with a higher percentage of successes.
Why Isolate cDNAs? The last topic to consider in this section is the question of why isolation of cDNAs is such a powerful approach.
A number of answers quickly spring to mind. Isolating cDNAs allows the experimentalist to use the cDNA to develop expression vectors so proteins of interest can be produced in high quantities, greatly simplifying the task of protein purification e. Knowing the sequence of an amino acid immediately gives access to the sequence of the protein.
Presence of sequences can easily suggest the protein product may be phosphorylated or may bind a particular small biochemical molecule, like GTP. The availability of a mRNA allows one to quickly design assays for studying the expression of the mRNA; labeling cDNA can be used to determine the expression of mRNA using both northern analysis and RNase protection and the subcellular distribution of RNase can be determined by in situ hybridization.
Each of these approaches provides a specific value. It reveals whether there are several different mRNAs that are expressed. Northern analysis relies on the fractionation of isolated mRNAs on agarose or occasionally, acrylamide gels which are then transferred to nitrocellulose.
In contrast to northern analysis, RNase protection relies on the resistance of a hybrid molecule to digestion. Whereas mRNA is normally extremely sensitive to ribonuclease, if RNA is isolated and then hybridized to a labeled probe either RNA or DNA then the hybrid molecule or a portion of a hybrid molecule can be protected from ribonuclease activity.
The amount of protected probe as well as its size can be easily measured. It is based on the hybridization to nucleic acids that are attached to a solid phase support. Knowing a cDNA's sequence, PCR primers can be designed and used to amplify a reverse transcriptase product although there are certainly problems in doing this quantitatively, it can also be a useful and powerful technique.
This approach can also be adapted to in situ approaches. The cDNA can also be used to determine which cells, tissues, or developmental stages produce a particular mRNA using in situ hybridization. Labeled nucleic acid is incubated with fixed tissue or cells under conditions where only specifically bound hybrid is stable.
Auto radiography reveals the position of endogenous RNA. As we have already noted above, knowledge of mRNA sequence can allow for the cloning of homologous sequences either from different species or additional members of the gene families within a species.
Availability of a cDNA makes production of both polyclonal and monoclonal antibodies much easier. Knowing the sequence of a protein allows one to design and synthesize a peptide that can be used as an antigen anti-peptide antigen.
Thus, in some cases, an antibody that recognizes a specific protein can be produced without ever purifying that protein. It also allows the expression and purification of a protein to be used as an antigen.
While expression vectors are extraordinarily useful in allowing production of large quantities of a protein, they are perhaps even more useful in that they allow production of not only a wild type protein but also production of a mutant protein. Coupled with site-directed mutagenesis it is possible to modify proteins to almost any end that the experimenter desires.
This allows tests of specific structure-function relationships. For example, the importance of a particular phosphorylation site in the activity of a protein or a specific residue in the binding of DNA can be studied by expressing mutant proteins. Of course, all such studies should be cognizant of the possibility that mutant proteins may be poorly expressed or unstable. More mundane uses of expressed proteins incorporating specific mutations include the production of specific proteins that can be used for biochemical reagents or biochemical products.
Would introduction of additional sulfhydral bonds increase the thermostability of a particular protein so it would be better for use in PCR or even better as a protease-based stain remover in laundry detergent? Isolation of cDNAs means that the in vivo function of a protein can be tested using a wide variety of approaches. A protein can either be overexpressed or its expression can be reduced or the function of a protein can be modified in a number of different ways.
Thus, the availability of cDNA clones brings many of the logical approaches of classical genetics to the molecular biologist and allows critical tests of in vivo function that would not otherwise be possible. The effort invested in isolating and characterizing a cDNA is well rewarded by the large number of uses that can be made of such a reagent. This can be done by northern analysis, by RNase protection assay, by PCR-based detection, or by in situ hybridization.
Label is generally incorporated into cDNAs by primer extension using a random selection of oligonucleotide hexemers. This technique has the ability to distinguish mRNAs of different molecular weights and so may reveal alternatively spliced products.
In this technique, a probe is generated by using a vector that incorporates a promoter for an RNA polymerase. This promoter can then transcribe in vitro a high specific activity RNA, part of which can be designed to be homologous to any cDNA. This synthesized RNA is of course extremely sensitive to ribonuclease treatment; however, if it is hybridized to a preparation of mRNA that includes a complementary sequence, a hybrid will be formed and this will render the RNA resistant to RNase digestion.
In some cases the amount of protected RNA can be measured directly but it can also be fractionated on gels to determine the molecular weight of the protected species. Again this is a sensitive method of detecting mRNA, but care is required to make quantitative claims about the amount of mRNA present in various samples. Finally, hybridization can be carried out in fixed tissues to determine what cell types express mRNA.
Again, mRNA can be detected by virtue of its hybridization with a labeled probe. Thus all of these methods have the ability to detect mRNA abundance and changes in mRNA among various cell types in response to development, and in response to hormones or other signaling molecules. The sequence of a mRNA is the quickest and most reliable way to identify the sequence of the encoded protein. With the rapidly expanding DNA database and the appreciation of how specific amino acid sequences can be used to define particular domains in proteins, the sequence information can be used extremely profitably.
For example, the sequence of a protein can be usedto determine the likelihood that particular regions of a protein will adopt an alpha-helix configuration.
Likewise particular sequences are associated with particular functions or particular structures. The zinc finger motif is a particular protein structure that can bind zinc atoms with high affinity and this structure is frequently found in DNA-binding proteins. Likewise, the helix-loop-helix structure which includes two alpha-helices connected by a loop, is frequently found in transcription factors. The catalytic triad is a sequence of 3 amino acids that is found in many proteases. Protein sequence will also reveal the presence of particular sites for post-translational modification.
The sequences for addition of carbohydrates, fatty acids, or phosphate groups are reasonably well conserved and the presence of these sequences is strong indicator about the post-translational modification of a protein. If alpha-helices are predicted and show a high concentration of hydrophobic groups on their surface, this is a strong indication that protein may have a transmembrane segment.
A repeated pattern of such a motif is found in many signaling molecules. For example, the classic seven transmembrane pattern that was originally found in bacterial rhodopsin is also present in many cell-surface receptors. Of course, any prediction made on the basis of amino acid sequence must be confirmed, but primary sequence is often a powerful indication of what experiments should be done.
The availability of a cDNA clone allows the protein to be expressed in a variety of contexts. A cDNA can be inserted into a variety of expression vectors for different purposes. Perhaps the most obvious use of such an approach is to drive expressions to extremely high levels. This produces a rich source of protein that considerably eases the difficulty of protein purification. This can make available abundant supplies of protein for physiological testing or use as a reagent.
A more striking use of expression system was in the ability to express mutant proteins. Since it is possible to mutate DNA sequences essentially at will, it is possible to express not only the wild type proteins but also related proteins that have particular mutations. These mutations, if well designed, can be used to test particular structure-function relationships within a protein.
They can determine whether a particular residue is important for catalytic activity or for association with another protein. In a related and more practical way, proteins can be modified for specific uses. One can incorporate disulfide bonds to increase the thermal stability of proteins that have industrial and commercial applications.
Reagents that are used in molecular applications can be modified so unwanted activities are suppressed. For example, nuclease activities can be dissociated from polymerase activities in DNA plolymerases. One of the most interesting examples of expressing mutant proteins can be found in the design of dominant negative mutant of a protein that can interfere with the activity of an endogenous protein.
For example, if it is possible to separate the DNA-binding domain and the RNA polymerase activating domain from a transcription factor, expression of the DNA-binding domain in the absence of the activating domain might be expected to interfere with the activity of the endogenous domain. Many proteins function a multimers, so expression of a mutant protein can frequently interfere with the activity of an endogenous protein by interfering with protein-protein dimerization.
This strategy has been extremely useful in study of specific transcription factors. Likewise, intracellular signaling requires sequential interactions of a series of proteins. Expression of a mutant protein that can interact with one member of the cascade but not the subsequent downstream members can interfere with the function of endogenous protein. This strategy has been used very profitably by making truncated mutants of receptors that express only the extracellular but not the intracellular domain of a protein or by expressing mutant version of ras or other GTP-binding proteins that transduce the signal within the cell.
The availability of mRNA sequence also opens the possibility of taking a genetic approach to understanding protein function. Analysis of such a cell or tissue can help establish the function of a protein in vivo.
Likewise, information about the sequence of a cDNA or the gene encoding it can be used to develop a strategy to disrupt or modify the gene encoding the c-DNA.
Using homologous recombination it is possible to either disrupt and eliminate expression of a gene or to force the expression of an altered gene product. It is also possible to study the effect of a forced expression of any gene product in any tissue of interest. By taking advantage of understanding a particular promoter elements discussed on another page that are required for the expression of a protein in a particular tissue at a particular time, it is possible to make a gene or a hybrid gene that expresses any protein of interest.
Thus, it is possible to determine the effect, for example of overexpressing a neuropeptide gene on neural development or the formation of a specific connection in the nervous system.
It is possible to determine whether the expression of a wild type or a dominant negative form of a protein can interfere with any developmental process or lead to the development of known diseases. As we noted above, it is also possible to take advantage of cDNA sequences to isolate homologous genes. Using either low stringency hybridization or a PCR approach based on knowledge of conserved regions of genes, it is frequently possible to identify additional genes that are members of the family and maybe biologically important in the absence of any knowledge of their function.
Knowing the cDNA sequence of a protein will frequently facilitate the development of antibodies and monoclonal antibodies. Most simply, an overexpressed protein can be purified and used as an antigen. It is essential to use only the minimal number of amplification cycles needed to obtain sufficient material for sequencing to avoid over-amplification of the libraries, which is a major source of bias in the results.
Because the blunt-end ligation is inefficient, short restriction-site linkers are first ligated to both ends. Bacteriophage vectors possess the following advantageous over plasmid vectors:. Creative Biogene is a US-based manufacturer and provider of genomics and proteomics products and services for academic and governmental research institutes, pharmaceutical and biotechnology industry.
Spinning down mRNA using density gradient centrifugation. Bacteriophage vectors possess the following advantageous over plasmid vectors: Are more desirable when a large number of recombinants are required for cloning low-abundant mRNAs as recombinant phages are produced by in vitro packaging.
What remains is the ds cDNA. The natural function of restriction enzymes in bacteria is to recognize specific restriction site sequences in phage DNA most often palindromic DNA sequences , hydrolyze it and thus avoid infection.
Restriction enzymes that make a scissors cut through the two strands of the double helix leaves blunt ends. If you mix two of double-stranded DNA fragments with the same sticky ends from different sources e. When a recombinant vector with its foreign DNA insert gets into a host cell, it can replicate many copies of itself, enough in fact for easy isolation and study.
They can be cut with a restriction enzyme at a suitable location, leaving those sticky ends. Therefore, it will be necessary to attach linkers to either end of the ds cDNAs.
Steps in the preparation of vector and ds cDNA for recombination are shown below. To prepare for recombination, a plasmid vector is digested with a restriction enzyme to open the DNA circle. Linkers are short, synthetic double-stranded DNA oligomers containing restriction sites recognized and cut by the same restriction enzyme as the plasmid.
Once the linkers are attached to the ends of the plasmid DNAs, they are digested with the appropriate restriction enzyme. This leaves both the ds cDNAs and the plasmid vectors with complementary sticky ends.
The next step is to mix the cut plasmids with the digested linker-cDNAs in just the right proportions so that the most of the cDNA linker ends will anneal form Hbonds with the most of the sticky plasmid ends. In early cloning experiments, an important consideration was to generate plasmids with only one copy of a given cDNA insert, rather than lots of re-ligated plasmids with no inserts or lots of plasmids with multiple inserts. Using betterengineered vector and linker combinations, this issue became less important.
They are added to E. Recall that transformation as defined by Griffith is bacterial uptake of foreign DNA leading to a genetic change. The transforming principle in cloning is the recombinant plasmid!
The transformation step is shown below. So when the recombinant cells are plated on agar, how do you tell which of the colonies that grow came from cells that took up a recombinant plasmid? Both the host strain of E.
One such plasmid vector carries an antibiotic resistance gene. In this case, ampicillin-sensitive cells would be transformed with recombinant plasmids containing the resistance gene. When these cells are plated on media containing ampicillin a form of penicillin , they grow, as illustrated below. Untransformed cells cells that failed to take up a plasmid lack the ampicillin resistance gene and thus, do not grow on ampicillin-medium. But, there is still a question.
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