Bacterial Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC) - PDF

Bacterial Artificial Chromosome Bacterial Artificial Chromosomes BAC vectors are plasmids constructed with the replication origin of E. coli F factor, and so can be maintained in a single copy per cell. These vectors can hold DNA fragments of up to 300 kb. Recombinant BACs are introduced into E. coli by electroportation. Once in the cell, the rBAC replicates like an F factor. 1) oriS, repE – F for plasmid replication and regulation of copy number. 2) parA and parB for maintaining low copy number and avoiding two F plasmids in a single cell during cell division. 3)A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection. 4) T7 and Sp6 phage promoters for transcription of inserted genes. The par genes, derived from F plasmid assist in the even distribution of plasmids to daughter cells during cell division and increase the likelihood of each daughter cell carrying one copy of the plasmid, even when few copies are present. The low number of copies is useful in cloning large fragments of DNA because it limits the opportunities for unwanted recombination reactions that can unpredictably alter large cloned DNA over time. Applications BAC vectors have been used in studying large double-stranded DNA viruses for academic research and d as a tool to develop advanced vaccines. BAC are utilized for the construction of genomic libraries. They have been used for basic science research to industrial research like animal husbandry. BAC are also used in determining phylogenetic lineage between different species. They are also used in the study of horizontal gene transfer. YAC Yeast Artificial Chromosome Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast. It is a human-engineered DNA molecule used to clone DNA sequences in yeast cells. Yeast artificial chromosomes (YACs) are plasmid shuttle vectors capable of replicating and being selected in common bacterial hosts such as Escherichia coli, as well as in the budding yeast Saccharomyces cerevisiae. Both yeast and bacterial cells can be used as hosts. The presence of ori/ARS from both bacteria and yeast respectively. 2. They are of relatively small size (approximately 12 kb) and of circular form when they are amplified or manipulated in E. 3. A large DNA insert of up to 200 - 500 kb can be cloned. 4. They are used for cloning inside eukaryotic cells. These act as eukaryotic chromosomes inside the host eukaryotic cell. It possesses the yeast telomere at each end. A yeast centromere sequence (CEN) is present which allows proper segregation during meiosis. 5. Many different yeast artificial chromosomes exist as ongoing refinements of the initial pYAC3 and pYAC4 plasmids constructed by scientist Burke. Process of Yeast Artificial Chromosomes YAC vector is initially propagated as circular plasmid inside bacterial host utilizing bacterial ori sequence. The circular plasmid is cut at a specific site using restriction enzymes to generate a linear chromosome with two telomere sites at terminals. The linear chromosome is again digested at a specific site with two arms with different selection marker. The genomic insert is then ligated into YAC vector using DNA ligase enzyme. The recombinant vectors are transformed into yeast cells and screened for the selection markers to obtain recombinant colonies. Telomeres helps in maintaiing chromosomal stability and prevent degradation by protecting the ends of chromosomes from DNA damage, recombination, and end-joining. Expression Vector Expression vector Expression vectors are vectors that enable the expression of cloned genes in order to determine the successful cloning process. Usually, cloning vectors do not allow the expression of a cloned gene which is why the use of expression vectors is required. The use of expression vectors facilitates the processing of introns in prokaryotes as these are designed with restriction sites next to the regulatory region. The restriction sites on the vectors result in splicing of the cloned gene to permit the expression of the gene under the regulatory system. The use of expression vectors is essential to determine the success of a cloning procedure and the efficiency of selective markers on the vectors. Expression vectors can be plasmid-based or viral- based that are introduced into the host cells in order to code for particular mRNAs. The expression vectors are often used for the production of proteins that can then be visualized by different methods depending on the complexity of the host cell. (SV40) SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that sometimes causes tumors in animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, leading to many early discoveries in eukaryotic DNA replication and transcription. Some vaccines made in the US between 1955 and 1961 were found to be contaminated with SV40, from the growth medium and from the original seed strain. Population level studies did not show extensive evidence of increase in cancer incidence as a result of exposure,though SV40 has been extensively studied. SV40 is a spherical virus with a circular, double stranded 5,243 bp chromosomes, which encodes 5 proteins, viz., small-t, large-t (both early protein), VP1, VP2 and VP3 (VP= virion protein),has an origin of replication(about 80 bp) and is complexed with histones to form chromatin. Large-T is essential for viral replication, while VP1, VP2 and VP3 form the viral capsid. SV 40 virus infects monkey kidney cell lines. The virus travels to the nucleus and gets uncoated. Then both the T-genes located near the origin are translated in the clockwise direction. The large T pro tein is important for virus DNA replica tion and starts after the translation of large T -protein. Replication starts at the origin and is bi-directional. It terminates when two replication forks meet. About 105 molecules of duplex DNA are synthesized per cell. Along with DNA replication, VP1, VP2 and VP3 proteins are synthe - sized. Then both the T-genes located near the origin are translated in the clockwise direction. The large T protein is important for virus DNA replication and starts after the translation of large T –protein. Advantages SV40 vectors (SV40) are good candidates for gene transfer, as they display some unique features. SV40 is a well-known virus, non replicative vectors are easy-to- make. They also efficiently transduce both, resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are nonimmunogenic. (i) it is easily modified to be non replicative. (ii) it can be produced in large quantities. (iii) it infects almost every cell type that has been tested, both dividing and quiescent. (iv) it is not immunogenic it allows long-term expression of the transgene. (v) its molecular biology is well studied. There are three types of SV 40 vehicles each of which have a distinct advantage or disadvantage among themselves. They are as follows: (a)SV40 Passive Transforming Vectors: These vectors neither replicate nor pro duce virions, but simply integrate the DNA segments into the cellular DNA. These transformed cells replicate the new DNA as an integral part of their own genomes. These plasmids are also shuttle vectors and include selective markers like herpes virus, thymidine kinase or neo genes. (b) SV40 Trans-ducting Vectors: These vectors are capable of replicating and packing into virion particles. Transducing vectors contain a segment of 300 bp which functions as the origin of replication and provides the transcriptional regulatory signals for the synthesis of mRNAs. This type of vector takes an insert of size 3.9 to 4.5 kb. These plasmids do not have the genes that code for VP1, VP2 and (c) SV40 Plasmid Vectors: These vectors multiply in the monkey cell line but are not packed as the virions. These vectors usually contain origin of replication sequences and larger T-protein gene but do not contain VP1, VP2 and VP3 genes. They are shuttle vectors, and have the ability to multiply both in E. coli and monkey cell line. What is Bioinformatics? Bioinformatics is an emerging branch of biological science that emerged from the combination of both biology and information technology. It is an interdisciplinary field of study that uses Biology, Chemistry, Mathematics, Statistics, and Computer Science that have merged to form a single discipline. This sector is mainly involved in analyzing biological data, and developing new software using biological tools. According to the NCBI- National Center for Biotechnology Information, the branch of NLM- National Library of Medicine and NIH- National Institutes of Health, Bioinformatics is defined as the analysis, collection, classification, manipulation, recovery, storage and visualization of all biological information using computation technology. The term Bioinformatics was first coined in the year 1960 by the two Dutch biologists named Paulien Hogeweg and Ben Hesper. According to their research and discoveries, Bioinformatics was defined as the study of information processes in biotic systems. Bioinformatics is defined as the application of tools of computation and analysis to the capture and interpretation of biological data. It is an interdisciplinary field, which harnesses computer science, mathematics, physics, and biology. Application of Bioinformatics Bioinformatics is mainly used to extract knowledge from biological data through the development of algorithms and software. Bioinformatics is widely applied in the examination of Genomics, Proteomics, 3D structure modelling of Proteins, Image analysis, Drug designing and a lot more. A significant application of bioinformatics can be found in the fields of precision and preventive medicines, which are mainly focused on developing measures to prevent, control and cure dreadful infectious diseases. The main aim of Bioinformatics is to increase the understanding of biological processes. Listed below are a few applications of Bioinformatics. In Gene therapy. In Evolutionary studies. In Microbial applications. In Prediction of Protein Structure. For the Storage and Retrieval of Data. In the field of medicine, used in the discovery of new drugs. In Biometrical Analysis for identification and access control for improvising crop management, crop production and pest control. Bioinformatic tools The main tools of a bioinformatician are computer software programs and the internet. A fundamental activity is sequence analysis of DNA and proteins using various programs and databases available on the world wide web. Anyone, from clinicians to molecular biologists, with access to the internet and relevant websites can now freely discover the composition of biological molecules such as nucleic acids and proteins by using basic bioinformatic tools. Biological Database A collection of biological data arranged in computer readable form that enhances the speed of search and retrieval and convenient to use is called biological database. A good database must have updated information. Types of Biological Databases Based on their contents, biological databases can be roughly divided into two categories 1. Primary databases Primary databases are also called as archieval database. They are populated with experimentally derived data such as nucleotide sequence, protein sequence or macromolecular structure. Experimental results are submitted directly into the database by researchers, and the data are essentially archival in nature. Once given a database accession number, the data in primary databases are never changed: they form part of the scientific record. Example : Protein Data Bank (PDB; coordinates of three- 2. Secondary databases Secondary databases comprise data derived from the results of analysing primary data. Secondary databases often draw upon information from numerous sources, including other databases (primary and secondary), controlled vocabularies and the scientific literature. They are highly curated, often using a complex combination of computational algorithms and manual analysis and interpretation to derive new knowledge from the public record of science. Example : UniProt Knowledgebase (sequence and functional information on proteins) There are also specialized databases that cater to particular research interests. For example, Flybase, HIV sequence database, and Ribosomal Database Project are databases that specialize in a particular organism or a particular type of data. Importance of Databases Databases act as a store house of information. Databases are used to store and organize data in such a way that information can be retrieved easily via a variety of search criteria. It allows knowledge discovery, which refers to the identification of connections between pieces of information that were not known when the information was first entered. This facilitates the discovery of new biological insights from raw data. Secondary databases have become the molecular biologist’s reference library over the past decade or so, providing a wealth of information on just about any gene or gene product that has been investigated by the research community. It helps to solve cases where many users want to access the same entries of data. Allows the indexing of data. It helps to remove redundancy of data. Protein sequence Database Universal protein sequence databases can be further subdivided into two categories: sequence repositories, in which data are stored with little or no manual inter- vention in the creation of the records; and expertly curated databases, in which the original data are enhanced by the addition of further information. Protein Sequence Databases The protein sequence database contains amino acid sequences of proteins and related information. The amino acid sequence of a protein is important because it determines the protein’s three-dimensional structure and function, as well as its identity. Some of the most popular protein sequence databases are: PIR PIR (Protein Information Resource) is a popular protein sequence database that provides information on functionally annotated protein sequences. SWISS-PROT SWISS-PROT is a protein sequence database that provides high levels of annotations, including information on the protein’s function, domain structure, post-translational modifications, and variants. Swiss-Prot is jointly managed by the SIB (Swiss Institute of Bioinformatics) and the EBI (European Bioinformatics Institute). The database distinguishes itself from other protein sequence databases by three criteria: (i) annotations, which cover a broad range of information, (ii) minimal redundancy, which ensures that each sequence is represented only once, and (iii) integration with other databases, which enables cross-referencing and retrieval of information from related databases. Protein Structure Databases Protein structure databases are collections of information related to the three-dimensional structure and secondary structure of proteins. There are several examples of protein structure databases. Some are: PDB PDB (Protein Data Bank) is a worldwide repository of 3D structure data on large molecules such as proteins, nucleic acids, and other biological macromolecules. It stores three-dimensional structural models of macromolecules obtained through three frequently used experimental methods: X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). SCOP SCOP (Structural Classification of Proteins) is a protein structure database that organizes proteins based on their secondary structure properties. SCOP categorizes proteins into different levels based on their evolutionary relationships and structural similarities. Proteins with high sequence identity or similar structure and function are grouped into families, and families with similar structures but low sequence identity are placed into superfamilies. Proteins with the same major secondary structures in the same arrangement are placed into the same fold category, and folds are further grouped into five structural classes. Applications of protein databases Protein databases have numerous applications. Some of the applications are: Protein databases can be used in sequence analysis to identify homologous sequences and predict protein functions based on sequence similarity. Protein databases can also be used for predicting protein structure by comparing the amino acid sequence of a protein with known structures in the database. Protein databases also include tools to study protein-protein interactions. Protein pattern and profile databases can be used for protein family identification by identifying conserved motifs. Protein databases such as metabolic pathway databases can be used in drug discovery and disease research by studying the metabolic pathways involved in diseases. Construction of Phylogenetic trees Building a phylogenetic tree requires four distinct steps: (Step 1) identify and acquire a set of homologous DNA or protein sequences, (Step 2) align those sequences, (Step 3) estimate a tree from the aligned sequences, and (Step 4) present that tree in such a way as to clearly convey the relevant information to others. What are the 3 types of phylogenetic tree? Distinct phylogenetic trees are divided into varied groups based on their different traits, such as whether they are rooted, non-rooted, bifurcating, or multifurcating. A phylogenetic tree is an estimate of the relationships among taxa (or sequences) and their hypothetical common ancestors. Today most phylogenetic trees are built from molecular data: DNA or protein sequences. Originally, the purpose of most molecular phylogenetic trees was to estimate the relationships among the species represented by those sequences. Phylogenetic trees represent hypotheses about the evolutionary relationships among a group of organisms. A phylogenetic tree may be built using morphological (body shape), biochemical, behavioral, or molecular features of species or other groups. In building a tree, we organize species into nested groups based on shared derived traits (traits different from those of the group's ancestor). The sequences of genes or proteins can be compared among species and used to build phylogenetic trees. Closely related species typically have few sequence differences, while less related species tend to have more. Seps for preparing the Phylogenetic Tree Selection of an organism or a gene family ↓ Selection of appropriate molecular markers ↓ Amplification ↓ Sequencing ↓ Assembly ↓ Alignment ↓ Evolutionary model ↓ Phylogenetic Analysis ↓ Construction of a Tree ↓ Evolution of a Phylogenetic Tree Importance of Phylogenetic Tree It is the fundamental tool to derive their most-useful evidence from the fields of anatomy, embryology, palaeontology and molecular genetics. Other significances of the phylogenetic tree are: 1.Used in the search for a new species. 2.Used to study evolutionary histories. 3.To study how the species were spread geographically. 4.To study the common ancestors of extant and extinct species. 5.It is used to identify the most recent common ancestors and to recognize how closely related species are. 6.To relate the milestones of the evolution of major life forms to the tree of life. 7.To represent evolutionary relationships between organisms that are believed to have some common ancestry. 8.With the help of the phylogenetic tree, the infectious microbes can be traced along with their evolutionary histories. Biotechnologies for ﬁrst-generation biofuels The plant varieties currently used for ﬁrst- generation biofuel production have been selected for agronomic traits relevant for food and/or feed production and not for characteristics that favour their use as feedstocks for biofuel production. Biotechnology can help to speed up the selection of varieties that are more suited to biofuel production – with increased biomass per hectare, increased content of oils (biodiesel crops) or fermentable sugars (ethanol crops), or improved processing characteristics that facilitate their conversion to biofuels. The ﬁeld of genomics – the study of all the genetic material of an organism (its genome) – is likely to play an increasingly important role. Genome sequences of several ﬁrst- generation feedstocks, such as maize, sorghum and soybean, are in the pipeline or have already been published. Apart from genomics, other biotechnologies that can be applied include marker-assisted selection and genetic modiﬁcation. Fermentation of sugars is central to the production of ethanol from biomass. However, the most commonly used industrial fermentation micro-organism, the yeast Saccharomyces cerevisiae, cannot directly ferment starchy material, such as maize starch. The biomass must ﬁrst be broken down (hydrolysed) to fermentable sugars using enzymes called amylases. Many of the current commercially available enzymes, including amylases, are produced using genetically modiﬁed micro- organisms. Research continues on developing efﬁcient genetic yeast strains that can produce the amylases themselves, so that the hydrolysis and fermentation steps can be combined. Application of biotechnologies for second-generation biofuels Lignocellulosic biomass consists mainly of lignin and the polysaccharides cellulose (consisting of hexose sugars) and hemicellulose (containing a mix of hexose and pentose sugars). Compared with the production of ethanol from ﬁrst-generation feedstocks, the use of lignocellulosic biomass is more complicated because the polysaccharides are more stable and the pentose sugars are not readily fermentable by Saccharomyces cerevisiae. In order to convert lignocellulosic biomass to biofuels the polysaccharides must ﬁrst be hydrolysed, or broken down, into simple sugars using either acid or enzymes. Several biotechnology-based approaches are being used to overcome such problems, including the development of strains of Saccharomyces cerevisiae that can ferment pentose sugars, the use of alternative yeast species that naturally ferment pentose sugars, and the engineering of enzymes that are able to break down cellulose and hemicellulose into simple sugars. Apart from agricultural, forestry and other by-products, the main source of lignocellulosic biomass for second- generation biofuels is likely to be from “dedicated biomass feedstocks”, such as certain perennial grass and forest tree species. Genomics, genetic modiﬁcation and other biotechnologies are all being investigated as tools to produce plants with desirable characteristics for second- generation biofuel production, for example plants that produce less lignin (a compound that cannot be fermented into liquid biofuel), that produce enzymes themselves for cellulose and/or lignin degradation, or that produce increased cellulose or overall biomass yields. METHODS OF DNA SEQUENCING CONTENT  CHEMICAL METHOD  ENZYMATIC METHOD  AUTOMATED DNA SEQUENCING CHEMICAL METHOD SUMMARY  Discovered by Maxam and Gilbert (1977)  This method of DNA sequencing involves chemical modification of DNA and subsequent cleavage at specific bases.  The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced.  Chemical treatment generates breaks at a small proportions of one or two of the four nucleotide based in each of four reactions (G,A+G, C, C+T). MAXAM AND GILBERT METHOD (CHEMICAL)  Thus a series of labelled fragments is generated, from the radiolabelled end to the first ‘cut’ site in each molecule.  The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation.  To visualize the fragments, the gel is exposed to X-ray film for autoradiography MAXAM AND GILBERT METHOD (CHEMICAL)  In this method following steps are involved:  (I) End labelling of DNA by 32P dATP  In this process the DNA fragment to be sequenced is end labelled by 32P dATP either at the 5’ end (by enzyme polynucleotide kinase) or at the 3’ end ( by deoxynucleotidyl transferase)  (II) Digestion or denaturation of end labelled fragment  Digestion: the end labelled fragment is digested with a restriction endonuclease which cleaves it into fragments  Denaturation: the end labelled fragment is denatured and is separated from its complementary strand through gel electrophoresis  (III) Base specific cleavage:  A) Modification of specific base by dimethyl sulphate or hydrazine  B) Removal modified bases from the DNA strand  C) Induction of strand break at the site from where the modified bases had been removed  Dimethyl sulphate adds a methyl residue to N7 position of G and N3 position of A  The methylation of G is about 5 times more frequent than A when DNA treated with dimethyl sulphate is heated, the methylated bases are lost from DNA strand and strand will break at the site of base loss  The breaks are 5 times more frequent at sites of G than at sites of A ( first cleavage product).  But when dimethyl sulphate treated DNA is subjected to acid treatment, methylated A is lost several times more frequently than methylated G  The break in DNA is more frequent at the site of A than G (second cleavage product)  Hydrazine acts on pyramidine under high salt concentration (NaCl)  It acts only on C (third cleavage product0  But otherwise, it acts equally well on C and T (fourth cleavage product)  Thus, under high salt concentration DNA breaks occur at the site where C is located while in the fourth mixture DNA break occurs at both C and T  Products from the four reaction mixture are subjected to gel electrophoresis in different lanes and the bands determined by autoradiography ENZYMATIC METHOD  Chain-terminator or dideoxy procedure for DNA sequencing was discovered by Fred Sanger in 1977  Depends on:  (i) Their ability to synthesize faithfully a complementary copy of a single-stranded DNA template  (ii) Their ability to use 2′, 3′-dideoxynucleotides as substrates the analog is incorporated at the growing point of the DNA chain, the 3′ end lacks a hydroxyl group and no longer is a substrate for chain elongation  Initiation of DNA synthesis requires a primer and usually this is a chemically synthesized  DNA synthesis is carried out in the presence of the four deoxynucleoside triphosphates, one or more of which is labeled with 32P and in four separate incubation mixes  In these, a low concentration of one each of the four dideoxynucleoside triphosphate analogs are added.  Therefore, in each reaction there is a population of partially synthesized radioactive DNA molecules, each having a common 5′ end, but each varying in length to a base-specific 3′ end.  After a suitable incubation period, the DNA in each mixture is denatured and electrophoresed in a sequencing gel DNA SEQUENCING WITH DIDEOXYNUCLEOSIDE TRIPHOSPHATES AS CHAIN TERMINATORS AUTOMATED DNA SEQUENCING  Fluorescent tags are attached to the chain-terminating nucleotides.  Each of the four dideoxynucleotides carries a spectrally different fluorophore.  The tag is incorporated into the DNA molecule by the DNA polymerase and accomplishes two operations in one step: it terminates synthesis and it attaches the fluorophore to the end of the molecule.  By using four different fluorescent dyes it is possible to electrophorese all four chain-terminating reactions together in one lane of a sequencing gel.  The DNA bands are detected by their fluorescence as they electrophorese past a detector  An alternative to the four-dye system is to start with a single fluorescent-labeled primer which is used in all four sequencing reactions.  The resulting fluorescent-labeled DNA strands are separated in four different lanes in the electrophoresis system Gene Therapy Gene therapy is a medical technique that treats or prevents disease by altering a person's genetic makeup. It can be used to treat both inherited and acquired disorders, such as hemophilia, sickle cell disease, and leukemia. Gene therapy works by:  Replacing a faulty gene: Replacing a disease-causing gene with a healthy copy  Inactivating a faulty gene: Turning off a disease-causing gene that's not functioning properly  Introducing a new gene: Adding a new or modified gene into the body to help treat a disease. First attempt at modifying human DNA was performed in 1980, by Martin Cline, but the first successful nuclear gene transfer in humans, approved by the National Institutes of Health, was performed in May 1989. One of the first scientists to report the successful direct incorporation of functional DNA into a mammalian cell was biochemist Dr. Lorraine Marquardt Kraus at the University of Tennessee Health Science Center in Memphis, Tennessee. Gene therapy may be classified into two types by the type of cell it affects: 1)somatic cell and 2) germline gene therapy. In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Any such modifications affect the individual patient only, and are not inherited by offspring. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia, and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy. In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations. In vivo method of gene transfer involves the transfer of cloned genes directly into the tissues of the patient.  This is done in case of tissues whose individual cells cannot be cultured in vitro in sufficient numbers (like brain cells) and/or where re-implantation of the cultured cells in the patient is not efficient.  Liposomes and certain viral vectors are employed for this purpose because of lack of any other mode of selection.  In case of viral vectors such type of cultured cells were often used which have been infected with the recombinant retrovirus in vitro to produce modified viral vectors regularly. These cultured cells will be called as vector-producing cells (VPCs)). The VPCs transfer the gene to surrounding disease cells.  The efficiency of gene transfer and expression determines the success of this approach, because of the lack of any way for selection and amplification of cells which take up and express the foreign gene. Vectors for GENE THERAPY : Vectors for gene therapy can be classified into two types: 1.Viral vectors : Adenovirus, Retrovirus, Adeno- Associated Virus, Lentivirus, Vaccinia virus, Herpes simplex virus 2. Non-viral : Physical methods – Electroporation, Gene Gun, Sonoporation, Magnetofection, Hydrodynamic delivery etc. Chemical methods – Oligonucleotides, Lipoplexes, Polymersomes, Polyplexes, Dendrimers, Inorganic Nanoparticles, Cell-penetrating peptides, etc Figure 1. Gene therapy using an adenovirus vector can be used to cure certain genetic diseases in which a person has a defective gene The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA (Fig). More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID). Patients with chronic liver disease and infection by the hepatitis virus which require a liver transplant, may be likely to undergo the hepatic transplantation of mature hepatocytes or those derived from iPS (Induced Pluropotent stem cells). Not only the transfer of genes might be needed to convert stem cells into hepatocytes; since the transplanted cells are susceptible to reinfection by the hepatitis virus, the transfer of a vector that encodes a short hairpin RNA directed against the virus would provide the transferred cells with resistance or ‘immunity’ to reinfection. Resistant cells can repopulate the liver over time and BASIC INTRODUCTION TO GENOMIC LIBRARIES INTRODUCTION It is necessary to obtain a very large number of recombinants, which together contain a complete collection of all of the DNA sequences in the entire genome, This is known as a genomic library. Although the human genome has been mapped, cloned, and sequenced in its entirety, it is still useful to examine how we might go about generating a genomic library and isolating a given gene, because the principles apply to all genomes. We could simply digest total genomic DNA with a restriction endonuclease, such as EcoRI, insert the fragments into a suitable phage λ vector, and then attempt to isolate the desired clone. How many recombinants would we have to screen in order to isolate the right one? There are two problems with the above approach. First, the gene may be cut internally one or more times by EcoRI so that it is not obtained as a single fragment. This is likely if the gene is large. Also, it may be desirable to obtain extensive regions ﬂanking the gene or whole gene clusters. Alternatively, the gene may be contained on an EcoRI fragment that is larger than the vector can accept. In this case the appropriate gene would not be cloned at all. These problems can be overcome by cloning random DNA fragments of a large size Since the DNA is randomly fragmented, there will be no systematic exclusion of any sequence. Furthermore, clones will overlap one another, allowing the sequence of very large genes to be assembled. Because of the larger size of each cloned DNA fragment, fewer clones are required for a complete or nearly complete library. GMO Genetically modified organism (GMO) It is a organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), including animals, plants, and microorganisms. In some genetic modifications, genes are transferred within the same species, across species (creating transgenic organisms), and even across kingdoms. Creating a genetically modified organism is a multi-step process. History Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973.They took a gene from a bacterium that provided resistance to the antibiotic kanamycin, inserted it into a plasmid and then induced other bacteria to incorporate the plasmid. The bacteria that had successfully incorporated the plasmid was then able to survive in the presence of kanamycin. The first genetically modified crop, an antibiotic-resistant tobacco plant, was produced in 1982. China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992. An insect resistant Potato was approved for release in the US in 1995, and by 1996 approval had been granted to commercially grow 8 transgenic crops and one flower crop. Bacteria were the first organisms to be genetically modified in the laboratory, due to the relative ease of modifying their chromosomes. Bacteria are cheap, easy to grow, clonal, multiply quickly and can be stored at −80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria, providing an unlimited supply for research. Left: Bacteria transformed with pGLO under ambient light Right: Bacteria transformed with pGLO visualized under ultraviolet light Plants Plants have been engineered for scientific research, to display new flower colors, deliver vaccines, and to create enhanced crops. Many plants are pluripotent, meaning that a single cell from a mature plant can be harvested and under the right conditions can develop into a new plant. This ability can be taken advantage of by genetic engineers. Some genetically modified plants are purely ornamental. They are modified for flower color, fragrance, flower shape and plant architecture. The most popular genetically modified organism, a blue rose (actually lavender or mauve) created in Suntory "blue" rose 2004. Crops Genetically modified crops are genetically modified plants that are used in agriculture. The first crops developed were used for animal or human food and provide resistance to certain pests, diseases, environmental conditions, spoilage or chemical treatments (e.g. resistance to a herbicide). The second generation of crops aimed to improve the quality, often by altering the nutrient profile. Third generation genetically modified crops could be used for non-food purposes, including the production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for Wild type peanut (top) and transgenic bioremediation. peanut with Bacillus thuringiensis gene added (bottom) exposed to cornstalk borer larva GM crops contribute by improving harvests through reducing insect pressure, increasing nutrient value and tolerating different abiotic stresses. Most currently available genes used to engineer insect resistance come from the Bacillus thuringiensis bacterium and code for delta endotoxins. A few use the genes that encode for vegetative insecticidal proteins.The only gene commercially used to provide insect protection that does not originate from B. thuringiensis is the Cowpea trypsin inhibitor (CpTI). CpTI was first approved for use cotton in 1999 and is currently undergoing trials in rice. Golden rice is the most well known GM crop that is aimed at increasing nutrient value. It has been engineered with three genes that biosynthesise beta-carotene, a precursor of vitamin A, in the edible parts of rice. It is intended to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A, a deficiency which each year is estimated to kill 670,000 Golden rice compared to white rice children under the age of 5 yeas. The original golden rice produced 1.6μg/g of the carotenoids, with further development increasing this 23 times. It gained its first approvals for use as food in 2018. Plants and plant cells have been genetically engineered for production of biopharmaceuticals in bioreactors, a process known as pharming. Biopharmaceuticals produced include cytokines, hormones, antibodies, enzymes and vaccines, most of which are accumulated in the plant seeds. Many drugs also contain natural plant ingredients and the pathways that lead to their production have been genetically altered or transferred to other plant species to produce greater volume. Therapeutics have been cultured in transgenic carrot and tobacco cells, including a drug treatment for Gaucher's disease. Genetically modified crops have been proposed as one of the ways to reduce farming-related CO2 emissions due to higher yield, reduced use of pesticides, reduced use of tractor fuel and no tillage. According to a 2021 study. Animals As of 2018 only three genetically modified animals have been approved, all in the USA. A goat and a chicken have been engineered to produce medicines. GM animals are created for research purposes, production of industrial or therapeutic products, agricultural uses, or improving their health. Mammals are the best models for human disease, making genetic engineered ones vital to the discovery and development of cures and treatments for many serious diseases. Knocking out genes responsible for human genetic disorders allows researchers to study the mechanism of the disease and to test possible cures. Genetically modified mice have been the most common mammals used in biomedical research, as they are cheap and easy to manipulate. Patenting And IPR Issues Patents are valuable tools that biotechnology startups can use to encourage investment and protect their intellectual property. The first thing that most people think of when they hear “intellectual property protection” is patents. Especially in the sciences, the prevalence of patents is understandable. The function of a patent is to ensure that an inventor has the right to protect his or her invention from others who would profit from it without permission. What Is a Patent? A patent is the granting of a property right by a sovereign authority to an inventor. This grant provides the inventor exclusive rights to the patented process, design, or invention for a designated period in exchange for a comprehensive disclosure of the invention. They are a form of incorporeal right. Government agencies typically handle and approve applications for patents. In the United States, the U.S. Patent and Trademark Office (USPTO), which is part of the Department of Commerce, handles applications and grants approvals. Types of Patents There are three types of patents available in the United States: utility patents, design patents, and plant patents. Each has its own specifications and durations. Utility Patents Utility patents, or patents for invention, issue legal protection to people who invent a new and useful process, an article of manufacture, a machine, or a composition of matter. Utility patents are the most common type of patent, with more than 90% of patents issued by the U.S. government belonging to this category.2 A utility patent lasts for 20 years from the date of filing as long as maintenance fees are paid. Maintenance fees are surcharges applied to utility patent applications filed after December 12, 1980 Design Patents Design patents are patents issued for original, new, and ornamental designs for manufactured products. Design patents protect the design or look of something. They require the invention to which the design belongs to be original and useful. Design patents last for 15 years for applications filed after May 13, 2015. For applications filed before May 13, 2015, patents last for 14 years from the date of the filing. Maintenance fees do not apply to design patents. Plant Patents Plant patents go to anyone who produces, discovers, and invents a new kind of plant capable of reproduction. These patents are granted for 20 years from the date of filing and no maintenance fees apply Patentable biotechnological inventions Methods for producing or analysing proteins and their use in an analysis method or in a medicinal product. Proteins, DNA sequences, microorganisms and constituents of the human body (for example, cells) which already exist in nature, if they are isolated from their natural environment or produced by a technical procedure, and have not been described previously. A gene, which is isolated and given a new task as a medicinal product or diagnostic tool. Genetically modified products, such as plants and animals. What is Intellectual Property? Intellectual property (IP) refers to creations of the mind, such as inventions; literary and artistic works; designs; and symbols, names and images used in commerce. IP is protected in law by, for example, patents, copyright and trademarks, which enable people to earn recognition or financial benefit from what they invent or create. By striking the right balance between the interests of innovators and the wider public interest, the IP system aims to foster an environment in which creativity and innovation can flourish. Intellectual property rights are customarily divided into two main areas: (i) Copyright and rights related to copyright. The rights of authors of literary and artistic works (such as books and other writings, musical compositions, paintings, sculpture, computer programs and films) are protected by copyright, for a minimum period of 50 years after the death of the author. Also protected through copyright and related (sometimes referred to as “neighbouring”) rights are the rights of performers (e.g. actors, singers and musicians), producers of phonograms (sound recordings) and broadcasting organizations. The main social purpose of protection of copyright and related rights is to encourage and reward creative work. (ii) Industrial property. Industrial property can usefully be divided into two main areas: One area can be characterized as the protection of distinctive signs, in particular trademarks (which distinguish the goods or services of one undertaking from those of other undertakings) and geographical indications (which identify a good as originating in a place where a given characteristic of the good is essentially attributable to its geographical origin). The protection of such distinctive signs aims to stimulate and ensure fair competition and to protect consumers, by enabling them to make informed choices between various goods and services. The protection may last indefinitely, provided the sign in question continues to be distinctive. Other types of industrial property are protected primarily to stimulate innovation, design and the creation of technology. In this category fall inventions (protected by patents), industrial designs and trade secrets. Ethical and Biosafety Issues The set of principles and standards to maintain the exploitation of the biological resources for the well-being of humans is called bioethics. Bioethics includes the ethical and moral issues in biology like modified crops, cloning, etc. The main aim of bioethics is to regulate the activity of biotechnology and human. There are many safety and ethical issues raised for GM crops and human cloning. Raising transgenic animals and plants has fueled ethical concerns, and the scientists have faced a lot of resistance where genetically modified crop plants or reproductive cloning research of human beings is involved. Thus, biosafety and bioethics are continuously being expanded to combine the rationale of ever- increasing scientific knowledge in biotechnology that is often in conflict with the long-standing social and moral value system of our society. Working with deadly disease-causing microorganisms for their characterization, diagnostics or therapeutics and vaccine development purposes are posing increasingly potential biosafety problems for laboratory workers. Thus, an appropriate biosafe working environment may protect workers from laboratory-induced infections. Polymerase chain reaction (PCR) CONTENT  Principle of PCR  Primer design  DNA Polymerases and Fidelity of thermostable enzymes principle  Polymerase chain reaction (PCR), which was discovered by Kary Mullis and for which he was awarded the Nobel prize in Chemistry in 1993. Principle  When a DNA duplex is heated, the strands separate or ‘melt’.  If the single stranded sequences can be copied by a DNA polymerase, the original DNA sequence is effectively duplicated.  If the process is repeated many times, there is an exponential increase in the number of copies of the starting sequence.  The length of the fragment is defined by the 5’ ends of the primers, which helps to ensure that a homogeneous population of DNA molecules is produced.  Thus, after relatively few cycles, the target sequence becomes greatly amplified, which generates enough of the sequence for identification and further processing POLYMERASE CHAIN REACTION (PCR) PRIMER DESIGN ASPECTS TO BE CONSIDERED IN PRIMER DESIGN  Source and sequence of the primer  If the primer sequence is known, it may be from the same gene from a different organism or may be from a cloned DNA  Synthesized oligonucleotide primers are commercially available  It may be derived from amino acid sequence data  In synthesizing the primer, two approaches can be taken.  By incorporating a mixture of bases at the wobble position, a mixed primer can be made, with the ‘correct’ sequence represented as a small proportion of the mixture  Alternatively, the base inosine (which pairs equally well with any of the other bases) can be incorporated as the third base in degenerate codons. PRIMER DESIGN  Primer length  The primer must be long enough to ensure that it is a unique sequence in the genome from which the target DNA is taken.  Primer lengths of around 20—30 nucleotides are usually sufficient  Repetitive sequences and regions of single-base sequence should be avoided  Should not contain regions of internal complementary sequence  Fidelity and stability  3’ termini (extension of PCR products occurs from here) of the primer is critical with respect to fidelity and stability of pairing with the target sequence.  Some ‘looseness’ of primer design can be accommodated at the 5 end DNA Polymerases and Fidelity of thermostable enzymes  DNA polymerase (Klenow fragment) is thermolabile and requires addition of fresh enzyme for each extension phase of the cycle  DNA polymerases isolated from hyperthermophilic organisms have become instrumental in overcoming these challenges  E.g. Taq polymerase (from Thermus aquaticus) and several versions of its recombinants as well as enzymes from Thermus flavus (Tfl polymerase) and Thermus thermophilus (Tth polymerase).  In addtion to Thermus-derived polymerases, a thermostable DNA polymerase from Pyrococcus furiosus is available.  This organism is classed as a hyperthermophile, with an optimal growth temperature of 1000C and is found in deep-sea vents.  Pfu polymerase is about 20 times more stable than Taq polymerase at 95°C  The key features required for a DNA polymerase include:  Processivity (affinity for the template, which determines the number of bases incorporated before dissociation),  Fidelity of incorporation  Rate of synthesis, and  The half-life of the enzyme at different temperatures.  Fidelity of incorporation of nucleotides is perhaps the most critical.  An error-prone enzyme will generate mutated versions of the target sequence out of proportion to the basal rate of misincorporation, given the repetitive cycling nature of the reaction.  The proofreading capability of a DNA polymerase defines fidelity, which increases the accuracy of DNA sequence replication.  High-fidelity DNA polymerases are enzymes with strong proofreading activity.  The ability of DNA polymerases to accurately replicate DNA sequences is crucial in applications such as cloning, sequencing, and site-directed mutagenesis TYPES OF PCR CONTENT  MULTIPLEX PCR  REVERSE TRANSCTION PCR  TOUCH DOWN PCR  NESTED PCR  HOT START PCR MULTIPLEX PCR  Multiplex PCR can be used to simultaneously screen for different pathogenic strains or isolates, or to differentiate pathogenic and non-pathogenic strains or isolates from each other using target specific primer pairs in combination  This gives us the ability to identify a pathogen or a group of pathogens in a population mixture containing other related or non-related organisms.  One important requirement is the specific selection of each primer set (to amplify a specific target) and the ability of the diagnostician to identify the pathogen type according to the size of amplicon or according to the sequence  A diagnostic assay that allows for the simultaneous detection of two or more pathogens is more cost effective and less time consuming than the implementation of multiple single assays for each pathogen.  A multiplex assay may be designed to allow for the detection of various closely related pathogens REVERSE TRANSCRIPTASE PCR  The starting template material is not DNA, but RNA.  Examples include PCR assays designed for the diagnosis of viral infections where the virus in question has a RNA genome, e.g. HIIV  Since RNA cannot serve as a template for PCR (RNA is not a substrate for the Taq DNA polymerases), reverse transcription is combined with PCR to make RNA into a complementary DNA  This conversion can be carried out in a simple enzymatic reaction with the enzyme reverse transcriptase (RT) and the DNA product then amplified by PCR.  In some cases PCR’s are specifically designed to amplify mRNA REVERSE TRANSCRIPTASE  This enzyme, used in the replication cycle of Retroviruses, is an RNA dependent DNA polymerase able to perform the following reactions:  Synthesizes a DNA strand on an RNA template  Removes the RNA strand from the DNA: RNA duplex (RNase H activity)  Synthesizes a second DNA strand on the DNA template  The most commonly of which are avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV) reverse transcriptase  Tth polymerase, an enzyme isolated from the bacterium Thermus thermophilus, is a heats table DNA polymerase with RT activity and can be used in PCR. CRITICAL FACTORS IN RT-PCR  Great care is needed as RNA is less stable than DNA  These enzymes are heat stable and difficult to get rid of  As general rule all water and buffers used in this process are treated with di-ethyl pyrocarbonate (DEPC) in a process that will destroy RNase  RT-PCR has found significant application in the field of disease diagnostics TOUCH DOWN PCR  In touchdown PCR, the PCR temperature cycle is started with an annealing temperature slightly higher than the calculated or anticipated annealing temperature of the primers.  A limited number of cycles with these conditions are carried out.  Thereafter the annealing temperature is lowered systematically for a limited number of cycles at each temperature profile  In a typical reaction, each of the PCR temperature cycles will differ by 1°C in the annealing temperature and each of these cycles is run twice  The rationale of this method is that preference is given to the reaction with the highest Tm (and therefore the highest specificity)  This method is useful in cases where the precise Tm of a primer is not known, or when you want to amplify multiplex amplicons using primer sets with different annealing temperatures  E.g. Use of Universal or common primers  The sequence of the primer is deduced from the amino acid sequence of the protein and the precise nucleotide sequence is thus not known  The touchdown method is therefore ideal to achieve amplification in cases where a number of mismatches between the primer and the template may occur  Note: Primer Melting Temperature (Tm) is the temperature at which one- half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability Nested PCR means that two pairs of PCR primers are used for a single locus. The first pair amplifies the locus as seen in any PCR NESTED PCR experiment. The second pair of primers (nested primers) binds within the first PCR product and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified by the specific internal primers. Step 2: 1st PCR reaction to Step 1: amplify the target DNA in Extraction of a general PCR using the outside PCR primer set target DNA. (red primers) Followed by the 2nd PCR reaction to amplify a fragment of the target DNA PROCEDURE in a specific PCR using the inside PCR primer set (blue primers). Step 3: Analysis of the PCR amplicons S IN A Step 4: Extraction of the amplicon DNA TYPICAL (band) from the gel, by agarose gel followed by DNA electrophoresis. sequencing NESTED PCR NESTED PCR HOT START PCR  Although primer sequence and length can be carefully designed to optimize its hybridization to only the intended target sequence at the annealing temperature, PCR amplification reactions can still have off-target amplification  Off-target amplifications are thought to occur during the lower temperature conditions of PCR sample preparation and thermal cycler ramping to the initial denaturation temperature.  Under these less stringent conditions, the primers can bind nonspecifically to regions of the nucleic acid target with partial complementarity or to other primer molecules  These nonspecific primer complexes may initiate the synthesis of undesired ‘mis-priming’ and ‘primer dimer’ extension products  This can compete with amplification of the desired target sequences  For improving the specificity of PCR is a Hot Start activation technique can be used.  The goal of this technique is to prevent the DNA polymerase from premature extension of primer complexes with lesser degrees of complementarity during the low stringency conditions of pre-PCR sample preparation.  In Hot Start activation, primer extension is blocked until the reaction mixture reaches an elevated, Hot Start temperature, where the stringency of the primer/target hybridization is optimal for specificity, and primer complexes are dissociated. OTHER APPROACHES INCLUDE REVERSE TRANSCRIPTASE AND cDNA SYNTHESIS INTRODUCTION: APPROACH FOR CLONING  Although the DNA represents the complete genome of the organism, it may contain non-coding DNA such as introns, control regions, and repetitive sequences.  This can sometimes present problems, particularly if the genome is large and the aim is to isolate a single-copy gene.  Messenger RNA has two advantages over genomic DNA as a source material.  First, it represents the transcriptome (i.e. the genetic information that is being expressed by the particular cell type from which it is prepared).  A second advantage of mRNA is that it, by deﬁnition, represents the coding sequence of the gene, with any introns having been removed during RNA processing.  Thus, production of recombinant protein is much more straightforward if a clone of the mRNA is available.  Sometimes the use of DNA, however, is inevitable.  Having decided on the source material, the next step is to choose the type of host/vector system.  Which host to chose?  For example when choosing among the wide variety of E. coli strains optimum host/vector combination should be ensured  When choosing a vector two things to keep in mind:  The method of joining the DNA fragments to the vector  The means of getting the recombinant molecules into the host cell CLONING FROM mRNA  Each type of cell in a multicellular organism will produce a range of mRNA molecules.  In addition to the expression of general housekeeping genes whose products are required for basic cellular metabolism, cells exhibit tissue- specific gene expression.  Thus, liver cells, kidney cells, skin cells, etc. will each synthesise a different spectrum of tissue-speciﬁc proteins (the proteome).  This requires expression of a particular subset of genes in the genome, achieved by synthesis of a set of mRNAs (the transcriptome).  In addition to the diversity of mRNAs produced by each cell type, there may well be different abundance classes of particular mRNAs.  This has important consequences for cloning from mRNA, as it is easier to isolate a speciﬁc cloned sequence if it is present as a high proportion of the starting mRNA population. SYNTHESIS OF cDNA  Complementary DNA (cDNA) is a DNA copy of a messenger RNA (mRNA) molecule produced by reverse transcriptase, a DNA polymerase that can use either DNA or RNA as a template.  It is not possible to clone mRNA directly, so it has to be converted into DNA before being inserted into a suitable vector.  This is achieved using the enzyme reverse transcriptase to produce complementary DNA (also known as copy DNA or cDNA).  The classic early method of cDNA synthesis utilizes the poly(A) tract at the 3’ end of the mRNA to bind an oligo(dT) primer, which provides the 3’-OH group required by RTase.  Given the four dNTPs and suitable conditions, RTase will synthesise a copy of the mRNA to produce a cDNA·mRNA hybrid.  The mRNA can be removed by alkaline hydrolysis and the single-stranded (ss) cDNA converted into doublestranded (ds) cDNA by using a DNA polymerase.  In this second-strand synthesis the priming 3’-OH is generated by short hairpin loop regions that form at the end of the ss cDNA.  After second-strand synthesis, the ds cDNA can be trimmed by S1 nuclease to give a ﬂush-ended molecule, which can then be cloned in a suitable vector. Synthesis of cDNA. Poly(A)+ RNA (mRNA) is used as the starting material. DRAWBACKS  Synthesis of full-length cDNAs may be inefﬁcient, particularly if the mRNA is relatively long.  This is a serious problem if expression of the cDNA is required, as it may not contain the entire coding sequence of the gene.  Such inefﬁcient full length cDNA synthesis also means that the 3 regions of the mRNA tend to be over-represented in the cDNA population.  Problems can arise from the use of S1 nuclease, which may remove some important 5 sequences when it is used to trim the ds cDNA.  More recent methods for cDNA synthesis overcome these problems to a great extent  One of the simplest adaptations involves the use of oligo(dC) tailing to permit oligo(dG)-primed second- strand cDNA synthesis.  The dC tails are added to the 3 termini of the cDNA using the enzyme terminal transferase. PHAGES  Cloning cDNA in phage (λ) vectors is, in principle, no different than cloning any other piece of DNA.  However, the vector has to be chosen carefully, as cDNA cloning has slightly different requirements than genomic DNA cloning in vectors  Generally cDNAs will be mu insertion vector has usually a large enough cloning capacity.  Short cDNAs that may not be representative full-length copies of the mRNA are removed.  In the case of many vectors cDNA is usually ligated into the EcoRI site using linkers  The recombinant DNA is packaged in vitro and plated on a suitable host for selection and screening. CLONING FROM GENOMIC DNA  If elements such as control sequences or introns are being investigated, or if genome sequencing is the goal, mRNA cannot be used (for cDNA cloning) and, thus, genomic DNA has to be isolated. SCREENING AND SELECTION OF RECOMBINANTS BLUE WHITE SCREENING Lac Z encodes β- galactosidase β-galactosidase converts X- Gal (colourless) to blue compound (X-Gal: 5-bromo-4-chloro-3- indolyl-β D- galactopyranoside) If a vector contains Lac Z gene, inserting DNA fragments into sequence encoding lac Z leads to insertional inactivation β-galactosidase is no longer produced. Therefore screening can be done to differentiate the recombinant and non-recombinant cells: Case 1: If a foreign gene is not taken up by the vector- non-recombinant cells Foreign gene is not inserted into LacZ gene. So Lac Z gene produces β-galactosidase. Therefore, on addition of substrate X-gal the enzyme β-galactosidase hydrolyzes X-gal to produce blue coloured compound. Colonies appear blue in colour. Case 2: If a foreign gene is taken up by the vector – recombinant cells Foreign gene is inserted into LacZ gene. The LacZ gene is disrupted so β-galactosidase is not produced. Therefore on addition of substrate X-gal no colour change is seen. Colonies appear white in colour. COLONY HYBRIDIZATION Colony hybridization is a screening method used in the selection of bacterial colonies with a desired DNA sequence. It is also called colony blot hybridization, colony lift or replica plating. It allows the screening of colonies plated with high density. The original procedure of colony hybridization was first developed by Grunstein and Hogness. First, the bacterial colonies can be transformed onto a membrane and the bacterial colonies are lysed, exposing the nucleic acids. Then, the exposed nucleic acids are fixed onto the membrane by denaturing them and are hybridized with the radioactive probes. A colony is a cluster of bacteria developed from a single bacterial cell through the asexual reproduction. Hence, all bacterial cells in a particular colony possess the same genetic makeup as well as the transformed genetic material. Generally, bacteria are transformed with the aid of plasmid or cosmid vectors. COLONY HYBRIDIZATION METHOD PLAQUE HYBRIDIZATION Plaque hybridization is the screening method for recombinant phages. It is a modification of colony hybridization by Benton and Devis in 1977. The method is also called plaque lift. Plaque hybridization procedure is similar to the colony hybridization procedure and the plaques are lifted by contacting them to a nitrocellulose membrane. Nucleic acids are then exposed and fixed on to the membrane, hybridizing with desired probes. A plaque is a clear zone on an agar plate produced by a particular bacteriophage by the lysis of bacterial cells on that area. IMMUNOCHEMICAL METHODS OF SCREENING Instead of radiolabeling DNA molecules, antibodies are used to identify the colonies developed that synthesize antigens encoded by the foreign gene/DNA present in the plasmids of bacterial colonies TOUCHDOWN PCR Touch-down PCR? In the touchdown PCR, “By sequentially decreasing the annealing temperature during each PCR cycle, the chance of the non-specific binding can be reduced.” Every PCR technique is evolved to eliminate unwanted amplification during the PCR reaction. The unwanted amplification, a non- specific binding results in false-positive or false-negative results. Also, the unwanted primer-dimer amplification results in the shorter non- specific bands which makes the primers INTRODUC unavailable for the target amplification. TION Primer-dimers and non-specific binding are two major setbacks of any PCR reaction or we can say it is a kind of curse for any PCR reaction. Non-Specific bindings are the amplicon other than the target sequence amplification. Hence it is necessary to stop any unwanted amplification in PCR reaction. By increasing the annealing temperature, the primers cannot bind to other than its complementary sequence and result in specific amplification, however, it is not always the case. So what to do to avoid unwanted amplification and get success in the PCR reaction? What is touch-down PCR? The touchdown PCR is the modification of the conventional PCR in which the high specificity of amplification is achieved by reducing the unwanted amplification on sequentially decreasing the annealing temperature after each PCR cycle. Mechanism of how the primers anneal to the PCR template DNA: The primers are the set of the short single-stranded DNA used in the PCR reaction which facilitates the amplification by providing the free 3’- OH ends to Taq DNA polymerase. For that, we have to design our target sequence-specific primers by using online tools. The primer must be between 18 to 22 nucleotides long, having 50% to 55% GC content and cannot form the primer dimers. The annealing temperature of the primers must be between 55°C to 65°C (ideally). The annealing temperature of the primers is the temperature at which the primers bind to their specific complementary sequence on DNA. Whereas, the melting temperature is the temperature at which the primer dissociates from the complementary DNA sequence. Also, the melting temperature is defined as the temperature at which half of the DNA strand is dissociated or denatured. Both definitions are right. So ideally, the annealing temperature should be lower than the melting temperature, and then the primers can bind to its complementary sequence. Generally, the annealing temperature is 5°C lower than the melting temperature but it is just an approximate assumption. Hence it might possible that at a given annealing temperature the primer may bind to the sequence other than its complementary sequences. It is hard to calculate the exact annealing temperature for impossible templates such as high GC-rich DNA. All these problems result in non-specific unwanted amplification. To overcome this problem, the touchdown PCR is designed. Principle of touchdown PCR In the touchdown PCR, the annealing temperature is selected 10°C higher than the melting temperature. Therefore primers can bind to only their specific and complementary sequence. After each cycle, the temperature is decreased by 1°C. Even if the DNA is not amplified in the initial cycles due to a higher temperature (temperature higher than the melting temperature), it will be amplified in the successive cycles, because we are decreasing the temperature after each cycle. Even if we are decreasing the temperature, non-specific bindings are not formed after the amplification of the first template. Once the DNA sequence of our interest is amplified successfully, that amplified DNA is work as a template for further PCR cycles. Graphical representation of sequential decrease of annealing in the touchdown PCR Advantages of touchdown PCR The touchdown PCR reduces the primer-dimer formation capacity of primers. It will also provide higher specificity by reducing the non-specific and unwanted bindings of the primer to the template DNA. The technique is extremely useful in the templates having higher GC contents. It also saves the time of the reaction.

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