Applications in Gene Selection and Cloning - Report Example

Recombinant DNA Technology: Applications in Gene Selection and Cloning Genetics and molecular biology is a relatively new field of biology, only having been recognized after the discovery of the double helix structure in the 1950’s. This is in contrast to other biological fields such as taxonomy or zoology. However, the last half of the 20th century has seen exponential growth in the field, with the creation of numerous applications of the knowledge about the basic structure and components of DNA. One of the most well-known and controversial uses of information from genetics and molecular biology is recombinant DNA technology. It is the process in which identified DNA sequences or amplicons are inserted into vectors, mostly circular DNA from bacteria, and are transported or inserted into host cells such as bacterial cells. Then, after several divisions, the number of cells increases, the new cells now known as recombinants carrying the inserted DNA sequence (Brown 2010). The basic process of inserting known gene sequences into vectors and cloning these using a variety of host cells has many applications that have a wide range of uses, from medicine to agriculture and even in the identification of relationships among organisms. The Construction of Recombinant DNA The possibility of inserting non-native DNA sequences into certain organisms was initially discovered by Salvador Luria and associates while studying why some phages were unable to grow in bacterial hosts while some were able penetrate host cells and integrate their viral DNA into the host (Allison 2009). Luria and associates also observed that the inserted phage DNA became permanently lodged in the bacterial host cell DNA, suggesting that it is possible to insert the genome of one bacterial strain to another via phage-mediated gene transfer (Berg and Mertz 2010). Other subsequent studies were able to show why only certain sequences were able to insert properly into host DNA, and were done through the use of enzymes identifying certain sites within the DNA where cuts can be made for the insertion of genetic material, later called restriction sites. These restriction sites are unique DNA sequences having 4-8 nucleic base pair sequence motifs that are identified by restriction endonucleases and cleaved, leaving the ends having single base pairs called sticky ends (Runge and Patterson 2005). These sticky ends become sites where foreign or native DNA sequences having the complementary base pairings could join onto, allowing for the insertion of a new set of genes into the sequence. Endonucleases are not limited to the creation of sticky ends, but are also used in cutting certain genetic sequences to be used for gene cloning or amplifications. These genetic sequences are amplified to check for purity or errors before being inserted in different types of host cells that allow the expression of the targeted amplicons (Figure 1) (Kieleczawa 2005). During the course of the advancement of molecular biology, several applications of recombinant DNA technology using these basic steps have been done, with most experiments focusing on using prokaryotic organisms as host cells. Studies that aim to identify the importance of certain genetic sequences in living systems use recombinant DNA technology to create mutants that either have or do not have the identified amplicons. Many bacteria such as Escherichia coli release substances onto the surroundings in order to regulate cellular processes during stressful conditions, and this is accomplished through molecular exporters that release amino acids such as alanine or lysine (Hori et al. 2011). Recombinant mutants that contain defective genes that could not transcribe for sequences to create necessary exporter molecules end up accumulating a large amount of L-alanine within the cells, while those that have genes, which overexpress the export genes, have enhanced export rates of the amino acid in comparison to the original parental strain. The study used recombinant plasmids as vectors for the cells to express mutant cells with enhanced transporters while the deletion mutants were created using deletion plasmids containing BamHI fragments that code for restriction endonucleases that remove the exporter Figure 1. In recombinant-based cloning, identified coding sequences are inserted into plasmids containing homologous sequences according to host cells. (Kieleczawa 2005). Figure 2. Basic steps in gene cloning in host cells (Brown 2010). sequences (Hori et al. 2011). By studying both the expression and non-expression of exporter sequences through the use of recombinant DNA molecules, the researchers were able to identify the functions of previously unknown genes. Cloning DNA in Host Cells Propagating the vector inside host cells and letting these undergo cell division is in essence DNA cloning in small-scale. But if the vector does not have homologous sequences with the host cell’s DNA, it would be impossible for the vector to propagate and it would be labeled as foreign DNA, which would eventually be digested by enzymes called restriction endonucleases (Kieleczawa 2005). Their primary function is to remove foreign DNA within the cell. However in recombinant DNA technology these enzymes allow cutting of sequences in specific restriction sites. If these vectors contain amplicons with similar sequences to that of the host cells’ DNA, these would not get digested in the cells and can divide and multiply along with the host cells, which makes recombinant technology possible. Some vectors contain amplicons that do not cause any morphological changes to the host cell. But in some experiments, the amplicons contain certain sequences that cause some phenotypic characteristics to appear in the host cells, causing them to become transformed (Runge and Patterson 2005). These transformed cells carry on reproducing both the genetic sequence as well as phenotypic trait, forming colonies carrying similar DNA sequences and displaying similar traits, which can later be harvested for studies or for mass production of compounds such as antibiotics or enzymes in vivo (Berg and Mertz 2010). Figure 2 shows how amplicons are cloned within host cells. Figure 3. Various genes can be cloned in vivo at the same time using SLiCE, where a generic vector (PTXB1) could easily uptake any gene sequences available in the cell product suspension using electrophoresis (Zhang et al. 2012). Cells that become transformed could either have imbibed loose genetic material from their surroundings or became infected by bacteriophages, or simply phages. Phages were first discovered to have the potential to alter the genetic diversity of bacteria by their nature of being able to penetrate the host’s cellular membrane and insert viral coding sequences into host DNA (Rooks et al. 2010). This is one reason why these phages are also used to insert vectors into bacterial cells in vivo. However, recent studies use novel cloning methods such as SLiCE that rely less on the use of phages, and instead facilitate in vitro seamless cloning through the recombination of short-ended homologous sequences without the use of restriction endonucleases (Zhang et al. 2012). This is done through an efficient system that prevents the creation of unwanted sequences at restriction sites by avoiding the use of enzymes that modify the end insert sequences of the vectors such as T4 DNA polymerase. This allows the assembly of an increased variety of DNA fragments instead of single sequences, as used in older cloning methods. Figure 3 shows how SLiCE could clone several genes at the same time. Using living cells for recombinant hosts mean that many DNA pretreatments and post-treatment methods are needed for stability, which costs time and effort. This also creates sticky ends which loose nucleotide pairs could attach to, causing unnecessary sequences to add to the amplicon. But using SLiCE as a method of creating short DNA fragments in vitro simply relies on nucleotide bases available within bacterial extracts and not on living cells (Zhang et al. 2012). Sequences are transformed within the bacterial cell extracts through the use of electrophoresis, which increases the efficiency of gene amplification. This method of cloning DNA fragments can save both time and efforts as the targeted amplicons can easily be cloned in any vector and can be propagated using any kind of bacteria available in the laboratory, as well as not having to depend on largely on restriction sites. Use of Recombinant Libraries Cloned and sequenced DNA may be stored in systems to be used as reference samples for future uses. These repositories are called recombinant libraries, containing genetic clones or sequences that were isolated and identified from test organisms (Runge and Patterson 2005). These recombinant libraries are built according to the abundance of the mRNA coding for targeted sequences, thus genes that produce high amounts of gene products have higher chances to be included in these libraries, while rare genes could only be found in certain repositories (King 2000). Genomic libraries are larger versions of recombinant libraries, having larger sequences containing numerous genetic sequences or genes with high molecular weights. The purpose of creating recombinant or genomic libraries is to map out genetic information unique to individual species as well as to map out which sequences are homologous among different species. However, each of these clones does not completely represent the full genomic sequence of species, which could cause issues such as inadequate genomic representations and the occurrence of rare genetic sequences. Still, the use of recombinant libraries is an important part of creating recombinant DNA molecules by using genetic sequences with identified base-pairings and protein products (Hoogenboom 2005). Recombinant libraries are used in identifying homologous sequences present in related species. For example, calcium sensors identified in various cereals have around 600bp, a fact identified through cloning and sequencing as well as identifying homologous sequences in multiple samples (Nath et al. 2010). Based on the homology of the sequences, presence of certain amino acid residues, and the presence of a conserved calcium sensor region, the phylogenetic relationship of certain cereals such as millet, sorghum, rice, maize and wheat were established. In another example of the use of recombinant libraries, mutant libraries of bacteria such as E. coli are created using PCR amplification that increases the chances for errors or mutagenesis (Abou-Nader and Benedik 2010). This is used in order to find out which mutant lines contain or lack the desired characteristics or traits in question, thus allowing for the identification of important sequences as well as inserting these sequences to create the amount of gene products as needed. Using identified coding sequences for cell-controlled death, vectors can be created which could be selective of either toxic antitoxin genes. Strains which lack certain sequences would not be able to uptake vectors of either toxic or antitoxin genes, and gene sequences that code for cell death would digest these instead (Abou-Nader and Benedik 2010). On the other hand, strains that have sequences that have cell-death inhibitory effects would be able to proliferate as normal. By creating mutants through error-prone PCR, a mutant recombinant library can be created, and by testing specific genes for specific phenotypic expressions the effects of deleting each coding sequence can be established. Clone Sequence Analysis In order to investigate fully the totality of an organism’s active genes, the transcripts of all active genes within the organism are sequenced, collected, and stored in genome libraries (Matsumoto et al. 2011). Sequencing of these active genes is done in order to establish the genome of the organism, as well as to establish libraries containing each gene and its corresponding product. In order to identify redundant or non-coding genetic sequences, genomic clones are identified using homologous sequences from genomic libraries, which are kept and used as representative sequences for succeeding tests. Also, these clones are sequenced against other species’ genomic libraries with known full-length complimentary DNA’s to identify homologous sequences that can be further tested to check if these sequences produce similar gene products in the species in question (Matsumoto et al. 2011). The sequences in question can be analyzed in various ways, from clone analyzing kits that require a few samples to sequencing machines that can process multitudes of samples at the same time. Genomic clones are important components of recombinant DNA technology since it would be difficult to insert probes or primers when there are no similar sequences between the vector and the host cell (Allison 2009). In turn, protein sequences for coding genes can be established from these clones, such as important organism traits. The base-pair sequences of valuable characteristics among crops are identified through the use of clones and obtaining the base-pair sequences of each trait. For example, quality strains of barley are sequenced to create genomic resources for valuable cultivars (Matsumoto et al. 2011). By obtaining the full genome of particular strains of barley identified to have excellent brewing characteristics, other unidentified or highly-hybridized strains can be sequenced to check for clones similar to those that exist in the genome library of barley. Also, it would be easier to isolate which traits needed to be over-expressed or under-expressed, thus increasing the specificity of recombinants in producing the desired trait. This is one application of clone sequencing and analysis that is most used in recombinant technology. Comparative sequence analysis of clones is not limited to identifying desirable traits, but it is also used for identifying diseases and the presence of pathogens (Sekar et al. 2006). Bacterial species believed to cause pathogenesis among susceptible organisms such as corals can be identified based on cloned sequences and by comparing the percentage homology of these to existing clone libraries of identified pathogenic species. Also, symbiotic bacteria living among the coral colonies can be sequenced to identify whether or not these bacteria are capable of inducing coral infections such as black-band disease (BBD) or if these are capable of harboring the pathogenic organisms. Using clone sequences sequenced against known BBD clone libraries, dinoflagellate clone libraries, and sequences from symbiotic bacteria, researchers were able to identify which particular bacterial species induce the BBD among corals, and which do not cause the disease. Identifying clone sequences and analyzing homology was important in this case so as to identify which organisms could cause BBD in corals as well as to check if other organisms are capable of inducing the disease. Aside from disease detection and trait selection, another use of clone sequence analysis is in establishing phylogenetic relationships among large groups of understudied organisms such as bacteria. For example, the phylogenetic relationships of Epsilonprotobacteria found in various environments such as deep-sea vents, sulfur-rich surroundings and petroleum-contaminated groundwater were established using clone sequences (Engle et al. 2003). 16S Ribosomal RNA clones were used to establish which sequences were homologous between Epsilonprotobacteria and whether or not certain differences between the sequences would be present within the group. It was found out that bacterial samples from groundwater, sulfur springs and caves have 97-99% similarities with existing Epsilonprotobacteria clone libraries, while the bacteria collected from deep-sea vents contain only 90-94% similarity to available clone libraries. Results indicated that the deep-sea vent bacteria came from a different clave, as suggested by lesser percentages of homologous sequences. Still, the high rate of similarity to existing Epsilonprotobacteria shows the close relationship of these bacteria living under the sea with those found on land. Conclusions One of the most significant contributions of genetics and molecular biology is recombinant DNA technology, accomplished by adding amplicons or target gene sequences onto vectors that are then inserted into host cells, and transforming them in the process. Cell division of the transformed cells or recombinants allow for inserted genes to become duplicated or cloned, producing either numerous cells with the desired genes or mass-producing other products as a result of the expression of these genes. As a result of the insertion and/or expression of these foreign genes, the host cells become transformed cells, with recombinant DNA making these varied from the original parental cells. Recombinant DNA molecules are the results of combining DNA sequences that do not occur naturally in nature. Various uses of recombinants may range from mass-producing gene products to identifying gene sequences and the respective products. However, in order to increase the number of these sequences, transformed cells must continually divide and multiply in numbers to achieve the desired amount of recombinant material. This is usually done using living host cells, however some recent experiments were able to do this in vitro, thus it is suggested that further studies regarding in vitro cloning be done in the future. Cloned gene sequences that are successfully sequenced are usually saved as samples, and these identified sequences and their corresponding gene products are collected and placed in repositories called recombinant libraries. Molecular engineering using recombinant cells containing desired traits is not the only field that puts importance to these libraries, but also other various other fields such as taxonomy and pathology. Sequences for testing are checked against individual entries in each library, and the presence of homologous sequences between samples from the recombinant library and test samples indicate that the latter are genomic clones of samples from the former, thus establishing the identity of samples as those similar to or related to the original organism used in the recombinant library. Recombinant DNA technology has come a long way from its initial theoretical uses, and now it has more uses aside from identifying the percent homology of the genes of one organism to another. The method can also be used to identify which sequences code for desirable traits to enhance quality of organisms such as crops, which sequences must be deleted to decrease gene expression in an organism, to identify the presence of pathogenic organisms in samples, as well as to establish the degree of phylogenetic relationships among groups of organisms with numerous species. While at present, living host cells and restriction endonucleases are necessary in order to accomplish these uses of recombinant DNA, is expected that in the future there would be no need for living host cells or endonucleases and instead the mere presence of vectors, free nucleic acid bases and amplicons would be sufficient to create a mass of gene clones that can then be used to transform any kind of organism without too much vector pre- and post-treatment measures. References Abou-Nader M, Benedik M. 2010. Rapid generation of random mutant libraries. Bioengineered Bugs. 1(5): 337-340. Allison L. 2009. Fundamental molecular biology. Hoboken (NJ): John Wiley & Sons, Ltd. Berg P, Mertz J. 2010. Personal reflections on the origins and emergence of recombinant dna technology. Genetics. 184(1): 9–17. Brown T. 2010. Gene cloning and DNA analysis: an introduction. Hoboken (NJ): John Wiley & Sons Ltd. Engel A, Lee N, Porter N, Stern L, Bennett P, Wagner M. 2003. 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