Genetics PDF

GENETIC CODE AND ITS PROPERTIES Genetic code are the sequence of nucleotides or nitrogenous bases present on messenger RNA (m RNA). As they are originating from DNA their bases are complementary to the segment of DNA, from where they are originated. In our body there are 20 different types of amino...

GENETIC CODE AND ITS PROPERTIES Genetic code are the sequence of nucleotides or nitrogenous bases present on messenger RNA (m RNA). As they are originating from DNA their bases are complementary to the segment of DNA, from where they are originated. In our body there are 20 different types of amino acids but the number of nitrogenous bases on mRNA is just four (A,U,G and C). Among the 64 triplet codons 61 is sense codon, ie. they code for amino acids. There are three codons –UAA, UAG and UGA they do not code for any amino acid, known as tremination codon. Genetic code AUG is considered as the initiation codon being it is always present at the initiation point. AUG code for amino acid methionine. Properties of genetic code- Triplet- Three nitrogenous bases comprises one genetic code arranged in a specific sequence. (About this property we have discussed already in the above paragraph). Commaless- there is no comma or any punctuation mark between the adjacent genetic codes. Non-overlapping- On the mRNA, one codon do not overlap with the next codon. EgAUG GCA ACG GGA etc. Universality- Same genetic code is present in all organism including virus, bacteria and all living organism. Degeneracy of genetic code- as the number of genetic code is more than the number of amino acids, it is implide that one amino acid may be coded by more than one codon. This is known as degeneracy. To understand see the table above. Ambiguity- A genetic code is always nonambiguous, ie. One codon never code for two different amino acids. Collinearity- The codons on mRNA and the corresponding amino acid residues in the polypeptide chain have a linear arrangement. Transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. Bacterial transcription is governed by three main sequence elements: Promoters are elements of DNA that may bind RNA polymerase and other proteins for the successful initiation of transcription directly upstream of the gene. Operators recognize repressor proteins that bind to a stretch of DNA and inhibit the transcription of the gene.  Positive control elements that bind to DNA and incite higher levels of transcription An operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately In eukaryotes there are a number of additional mechanisms through which polymerase activity can be controlled. These mechanisms can be generally grouped into three main areas: Control over polymerase access to the gene. This includes the functions of histone remodeling enzymes, transcription factors, enhancers and repressors, and many other complexes. Productive elongation of the RNA transcript. Once polymerase is bound to a promoter, it requires another set of factors to allow it to escape the promoter complex and begin successfully transcribing RNA. Termination of the polymerase. A number of factors which have been found to control how and when termination occurs, which will dictate the fate of the RNA transcript. Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. Genetic manipulation is one of the biomedical technologies to replace a defective gene, correct a mutational gene, and induce an intrinsic healing potential. Genetic engineering could potentially fix severe genetic disorders in humans by replacing the defective gene with a functioning one. Drugs, vaccines and other products have been harvested from organisms engineered to produce them. Crops have been developed that aid food security by increasing yield, nutritional value and tolerance to environmental stresses. Genetic engineers must first choose what gene they wish to insert into the organism. The next step is to isolate the candidate gene. The cell containing the gene is opened and the DNA is purified. The gene is separated by using restriction enzymes to cut the DNA into fragments or polymerase chain reaction (PCR) to amplify up the gene segment. These segments can then be extracted through gel electrophoresis. Once isolated the gene is ligated into a plasmid that is then inserted into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited copies of the gene are available. Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a promoter and terminator region, which initiate and end transcription. Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome. Recombinant DNA is the general name for a piece of DNA that has been created by combining two or more fragments from different sources because DNA molecules from all organisms share the same chemical structure, differing only in the nucleotide sequence. DNA molecules can originate from any species. For example, plant DNA can be joined to bacterial DNA, or human DNA can be joined with fungal DNA. Molecular cloning is the laboratory process used to produce recombinant DNA. It is one of two most widely used methods, along with polymerase chain reaction (PCR), used to direct the replication of any specific DNA sequence chosen by the experimentalist. There are two fundamental differences between the methods. One is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. Recombinant DNA is widely used in biotechnology, medicine and research. Recombinant human insulin has almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of type 1 diabetes. Hepatitis B infection can be successfully controlled through the use of a recombinant subunit hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. Molecular cloning refers to the isolation of a DNA sequence from any species (often a gene), and its insertion into a vector for propagation, without alteration of the original DNA sequence. Once isolated, molecular clones can be used to generate many copies of the DNA for analysis of the gene sequence, and/or to express the resulting protein for the study or utilization of the protein’s function. The clones can also be manipulated and mutated in vitro to alter the expression and function of the protein. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. These are termed as inserts. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA.  Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMOs) Transgenic and Knockout Animals are animals into which additional and/or altered genetic information has been introduced. Two principle technical approaches are used to generate transgenic animals: (i) addition of genetic information via microinjection of foreign DNA into the pronucleus of zygotes. Termed as transgenic animals (ii) deletion or modification of an endogenous target gene. Termed as gene knockout animals. So far, gene targeting is applicable only in mice. Pronuclear microinjection is used in a wide variety of different species. Reverse transcription and cDNA Reverse transcription is the synthesis of DNA from an RNA template. This process is driven by RNA-dependent DNA polymerases, also known as reverse transcriptases. Reverse transcriptases occur naturally in both prokaryotic and eukaryotic organisms, as well as in retroviruses. cDNA (copy DNA; also called complementary DNA) is synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase. While DNA is composed of both coding and non-coding sequences, cDNA contains only coding sequences. Scientists often synthesize and use cDNA as a tool in gene cloning and other research experiments. cDNA is often used to express a specific protein in a cell that does not normally express that protein. Such DNA is than inserted in other organism e.g. yeast which is good tool to make lots of proteins. Reverse transcriptases have been identified in many organisms, including bacteria, animals, and plants, as well as viruses. Natrually, reverse transcription contributes to: Propagation of retroviruses—e.g., human immunodeficiency virus (HIV), Replication of chromosomal ends called telomeres. Restriction enzyme, restriction endonuclease, It is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system. More than 3,600 restriction endonucleases are known which represent over 250 different specificities. Over 3,000 of these have been studied in detail, and more than 800 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning Pallindrome sequence: In molecular biology, palindromic sequences are referred to as the sequence of nucleotides in the DNA duplex or RNA, where the sequence in one strand is the same as the complementary sequence of the other strand when read from the same direction on both the strands, either 5’ to 3’ or 3’ to 5’. 5′ —— GAATTC —— 3′ 3′ —— CTTAAG —— 5′ Many restriction enzymes or restriction endonucleases identify a specific palindrome site and cut the DNA strands. The human genome has many palindromic sequences distributed throughout. They play role in DNA replication, gene expression and regulation. Palindromic sequences account for major deletions and insertions during DNA replication. They also promote interchromosomal recombination and translocation. Shorter palindromic sequences (>50 bp long) provide stability to DNA but a longer palindromic sequence makes DNA vulnerable to mutations and makes it unstable. Polymorphism, is a discontinuous genetic variation resulting in the occurrence of several different forms or types of individuals among single species i.e., alternate phenotype. In simple words these are two or more possibilities of a trait on a gene. Since all polymorphism has a genetic basis, genetic polymorphism has a particular meaning i.e., Genetic polymorphism is the occurrence in the same population of two or more alleles at one locus, each with appreciable frequency", where the minimum frequency is typically taken as 1%. The most common type of polymorphism involves variation at a single nucleotide (also called a single-nucleotide polymorphism, or SNP). Other polymorphisms can be much larger, involving longer stretches of DNA like Small-scale insertions/deletions (Indels) consist of insertions or deletions of bases in DNA. The most obvious example of this is the separation of most higher organisms into male and female sexes. Another example is the different blood types in humans. The smooth graduation of height among individuals of human populations and the graduations possible between the different geographic races. If the frequency of two or more discontinuous forms within a species is too high to be explained by mutation, the variation—as well as the population displaying it—is said to be polymorphic. The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA. PCR consists of three major phases: denaturation, hybridization/annealing, elongation/amplification. During the denaturation phase, DNA is heated to 95 celsius (C) to dissociate the hydrogen bonds between complementary base pairs of the double-stranded DNA. Immediately following denaturation, the process of annealing occurs; annealing involves cooling the denatured DNA at a temperature ranging from 37-72 C allowing for the hydrogen bonds to reform. Annealing best occurs at temperatures between 55 C to 72 C. Annealing allows for the primers to bind to the single-stranded DNA at their respective complementary sites beginning at the 3’ end of the DNA template. Finally, an optimal reaction temperature, 75-80 C, that is best suitable for enzyme-induced DNA replication is selected to ensure DNA polymerase activity. This cycle repeats 25 to 35 times in a typical PCR reaction, which generally takes 2-4 hours, depending on the length of the DNA region being copied. Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments. DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may cause disease. Regions of DNA up to about 900 base pairs in length are routinely sequenced using a method called Sanger sequencing or the chain termination method.