Q.1 (i) A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearised by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. They were first described in 1983 by Murray & Szostak. (ii) Recombinant DNA is a form of synthetic DNA thereby combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered. Recombinant protein is protein that is derived from recombinant DNA. The Recombinant DNA technique was engineered by Stanley Norman Cohen in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine. (iii) The Basic Local Alignment Search Tool (BLAST) for comparing gene and protein sequences against others in public databases, now comes in several types including PSI-BLAST, PHI-BLAST, and BLAST 2 sequences. Specialized BLASTs are also available for human, microbial, malaria, and other genomes, as well as for vector contamination, immunoglobulins, and tentative human consensus sequences. (IV) Modification of Cut Ends – (True) Linker and/or adaptor molecules can be joined to the cut ends. Linkers are short, chemically synthesized, self complementary, double stranded oligonucleotides, which contain within them one or more restriction endonuclease sites, e.g., linker 5' -CCGAA TTC¬GG (only one strand of the linker is shown here) contains one EcoRI site. The 3'-ends of DNA strands always carry a free hydroxyl (-OH) group, while their 5'-ends always bear a phosphate group. Often the ends produced by restriction enzymes have to be modified for further manipulation of the fragments; some of the modifications are summarised below. 1. Removal of the 5'-phosphate group of vector DNA by alkaline phosphatase treatment in order to prevent vector circularization during DNA insert integration. 2. Addition of a phosphate group to a free 5'-hydroxyl group by T4 polynucleotide kinase. 3. Removal of the protruding ends by digestion with, say, S1 nuclease; this enzyme digests both 3'- and 5'-protruding ends. (v) Northern Blot (True) The northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. (vi) (True) In order for transcription to take place, the enzyme that synthesizes RNA, known as RNA polymerase, must attach to the DNA near a gene. Promoters contain specific DNA sequences and response elements which provide a binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase. • In bacteria, the promoter is recognized by RNA polymerase and an associated sigma factor, which in turn are brought to the promoter DNA by an activator protein binding to its own DNA sequence nearby. • In eukaryotes, the process is more complicated, and at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter. Promoters represent critical elements that can work in concert with other regulatory regions (enhancers, silencers, boundary elements/insulators) to direct the level of transcription of a given gene. (vii) Gene replacement is by point mutations but details are given below.TARGETED MUTAGENESIS AND GENE REPLACEMENT Gene targeting, targeted mutagenesis, or gene replacement: This powerful technology allows investigators to generate directed mutations at any cloned locus. These new mutant alleles can be passed through the germ line to produce an unlimited number of mutant offspring, and different mutations can be combined with variants at other loci to study gene interactions. (vii) Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell. Genes are regulated at either the transcriptional or post-transcriptional level. Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.). Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi. Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous genes. Mechanisms of gene silencing also protect the organism's genome from transposons and viruses. Gene silencing thus may be part of an ancient immune system protecting from such infectious DNA elements. Genes may be silenced by DNA methylation during meiosis, as in the filamentous fungus Neurospora crassa. (ix) RT PCR In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are: (1) the use of fluorescent dyes that intercalate with double-stranded DNA, and (2) modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. Frequently, real-time polymerase chain reaction is combined with reverse transcription to quantify messenger RNA (mRNA) in cells or tissues. Abbreviations used for real-time PCR methods vary widely and include RTQ-PCR, Q-PCR or qPCR.  Real-time reverse-transcription PCR is often denoted as qRT-PCR,, RRT-PCR, or RT-rt PCR. The acronym, RT-PCR, commonly denotes reverse-transcription PCR and not real-time PCR, but not all authors adhere to this convention. (x) (True) Introgression of genes from wild relatives is usually limited to the same species or genus (Harlan and Wet, 1971). The primary and secondary gene pools include individuals of the same species and those of other species with which gene transfer is possible even if difficult. In rice, interspecific hybrids between Oryza species may produce fertile plants (Naredo et al., 1997). The tertiary gene pool with more distant taxa that can not usually be accessed for genetic improvement may often contain genes of potential value. Hybrids reported between rice and Portersia coarcata, a more distant member of the Oryzeae tribe, recovered by embryo rescue were not fertile (Jenna, 1994). Here we provide a protocol for introduction of genes from the tertiary gene pool of rice (Oryza sativa) by microparticle bombardment with the unfractionated genomic DNA from a distant wild relative (Zizania palustris). This approach may be useful in expanding the gene pool of other economically important species. Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA from Zizania was introduced by this procedure. One plant had 16 of a possible 122 Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available. Q No 4. Recombinant DNA Technology Making Recombinant DNA (rDNA): An Overview • Treat DNA from both sources with the same restriction endonuclease (BamHI in this case). • BamHI cuts the same site on both molecules 5' GGATCC 3' 3' CCTAGG 5' • The ends of the cut have an overhanging piece of single-stranded DNA. • These are called "sticky ends" because they are able to base pair with any DNA molecule containing the complementary sticky end. • In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed. • DNA ligase covalently links the two into a molecule of recombinant DNA. Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5'GGCC3' 3'CCGG5' The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined. 3. Gene cloing vectors Vector" is an agent that can carry a DNA fragment into a host cell. If it is used for reproducing the DNA fragment, it is called a "cloning vector". If it is used for expressing certain gene in the DNA fragment, it is called an "expression vector". Commonly used vectors include plasmid, Lambda phage, cosmid and yeast artificial chromosome (YAC). Plasmid Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb. Plasmids Electron micrograph of an E. coli cell ruptured to release its DNA. The tangle is a portion of a single DNA molecule containing over 4.6 million base pairs encoding approximately 4,300 genes. The small circlets are plasmids. (Courtesy of Huntington Potter and David Dressler, Harvard Medical School.) Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They: • are small (a few thousand base pairs) • usually carry only one or a few genes • are circular • have a single origin of replication Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
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