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BCH4305 Biotechnology and Genetics Engin-1

The document provides an overview of DNA structure, replication, and the principles of biotechnology, emphasizing the role of nucleotides and base pairing in genetic information transfer. It discusses the historical development of biotechnology, its various branches, and applications in medicine, agriculture, and industry. Additionally, it outlines the central dogma of molecular biology, detailing the processes of transcription and translation in gene expression.

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51 views36 pages

BCH4305 Biotechnology and Genetics Engin-1

The document provides an overview of DNA structure, replication, and the principles of biotechnology, emphasizing the role of nucleotides and base pairing in genetic information transfer. It discusses the historical development of biotechnology, its various branches, and applications in medicine, agriculture, and industry. Additionally, it outlines the central dogma of molecular biology, detailing the processes of transcription and translation in gene expression.

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ibrahimaliibrahim6998
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF or read online on Scribd
“= PF | BCH 430% BIOTECHNOLOGY AND GENETIC ENGINEERING pxa (Deoxyribonucleic acid) “pNA isa group of molecules that is responsible for carrying and tra materials or the tenetic instructions from parents to offspring.” ADNA molecule comprises of two long polynucleotide chains composed of nucleotide subunits. Each of these chains is known as a DNA chain, or a DNA strand. Hydrogen bonds between the base portions of the ::ucleotides hold the two chains together. ‘A nucleotide is the basic building block of nucleic acids. RNA and DNA are polymers made of long chains of nucleotides. A nucleotide consists of a sugar molecule (cither ribose in RNA or deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base. iting the hereditary Seructure ofNucleotide] , A nitrogenous base is composed of carbon and nitrogen rings. The number of rings this base has determines whether the base is a purine (two rings) or pyrimidine (one ring). 11Page Scanned with |\CamScanner Purines: Pyrimiaines 4 are Na ‘ i ah H. is . Protein The term dogma means “set of beliefs”; it dates from the time thé idea was put forward first as a theory. Since then the “dogma” has been confirmed experimentally, but the term persists. ‘The central dogma is the vital principle of molecular genetics because it summarizes how the genetic information in DNA becomes expressed in the amino acid sequence in a polypeptide chain. ‘The sequence of nucleotides in a gene specifies the sequence of nucleotides in a molecule of messenger RNA; in tum, the sequence of nucleotides in the messenger RNA specifies the sequence ‘of amino acids in the polypeptide chain. jene Expressior ‘+ gene is a small section of DNA that contains the instructions for a specifie molecule, usually a protein. + The purpose of genes is to store information, Each gene ccutains the information required to build specific proteins needed in an organism. t * Gene expression is the process through which information from a gene is used in the synthesis of a functional gene product. These prodvets are often proteins, but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA. The process of gene expression involves two main stages: ‘Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase, and the processing of the resulting mRNA molecule, Translation: the use of mRNA to direct protein synthesis, and the subsequent post-translational processing of the protein molecule. ‘Some genes are r:sponsible for the production of other forms of RNA that play arole in translation, including transfer RNA (tRNA) and ribosomal RNA (rRNA). ‘The main concept in the central dogma is that DNA does not code for protein directly but rather acts through an intermediary molecule of ribonucleic acid (RNA). The structure of RNA is similar to, but not identical with, that of DNA. There is a difference in the sugar (RNA contains the sugar ribose instead of deoxyribose), RNA is usually single-stranded (not a duplex), and RNA [Page : ae Scanned with @ camscanner contains the base uracil (U) instead of thymine (T), which is present in DNA. Actually, three types of RNA take part in the synthesis of proteins: A molecule of messenger RNA (mRNA), which carries the genetic information from DNA and is used as a template for polypeptide synthesis. In most mRNA molecules, there is a high proportion of nucleotides that actually code for amino acids. Several types of ribosomal RNA (rRNA), which are major constituents of the cellular particles called ribosomes on which polypeptide synthesis takes place. A set of transfer RNA (tRNA) molecules, each of which carries a particular amino acid as well as a three-base recognition region that base-pairs with a group of three adjacent bases in the mRNA. As each tRNA participates in translation, its amino acid becomes the terminal subunit added tothe length of the growing polypeptide chain. The tRNA that carries methionine is denoted tRNAMet, that which carries serine is denoted tRNASer, and so forth. Gene control regio F + Start site. A start site for transcription. ‘A promoter. A region of few hundred nucleotides ‘upstream’ of the gene (toward the 5* end). It is no* transcribed into mRNA, but plays a role in controlling the transcription of the gene. Transcription factors bind to specific nucleotide sequences in the promoter region and assist in the binding of RNA polymerases. eukary 7 seque q ognized by on e i wing other transcrotion f tual . Babanests ‘Some transcription factors (called activators) bind to regions called ‘enhancers’ increase the rate of transcription. These sites may be thousands of nucleotides from tec ‘sequences or within an intron. Some enhancers are conditional and only work in the pres znce of other factors as well as transcription factors. + Silencers. Some transcription factors (called repressors) bind to, regions called ‘silencers’ that depress the rate of transcription. TRANSCRIPTION i : The process of making an RNA ‘strand from a DNA template is transcription, and the RNA ‘molecule that is made is the transcript. The manner in which genetic information is transferred iz|Page Scanned with @ camscanner from DNA to RNA. The DNA opens up, and one of the strands is used as a template for the synthesis of a complementary strand of RNA. The base sequence in the RNA is complementary (in the Watson-Crick pairing sense) to that in the DNA template, except that U (which pairs with A) is present in the RNA in place of T To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region called the promoter. Basically, the promoter tells the polymerase where to "sit down" on the DNA and begin transcribing Recognition ites Gia aces { oe Cer ETE Gun L § i ‘ a Sopp ee, own ee Senghe-stronded Sempote ‘ . “Bach gene (or, in bacteria, each group of genes transcribed together) has its own promoter. A promoter contains DNA sequences that let RNA polymerase or its helper proteins attach to the DNA. Once the transcription bubble has formed, the polymerase ean start transcribing Elongation Once RNA polymerase is in position at the promoter, the next step of transcription—elongation— can begin. Basically, elongation is the stage when the RNA strand gets longer, thanks to the addition of new fiucleotides. During elongation, RNA polymerase “walks” along one strand of DNA, known as the template strand, in the 3!o 5' direction. For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the 3' end of the RNA strand. The RNA strand looks similar to DNA, except that it contains the base uracil in place of thymine and has ribose sugars (which have a hydroxyl group on the 2' carbon) in place of deoxyribose sugars. . ! 13 [Page Scanned with @ camscanner RNA polymerase synthesizes an RNA transcript complementary to the DNA template strand in the S' to 3* direction. It moves forward along the template strand in the 3* to 5' direction, opening the DNA double helix as it goes. The synthesized RNA only remains bound to the template strand for a short while, then exits the polymerase as a dangling string, allowing the DNA to close back up and form a double helix. ‘The RNA transcript is nearly identical to the non-template, or coding, strand of DNA. However, RNA strands have the base uracil (U) in place of thymine (7), as well asa slightly different sugar in the nucleotide. So, as we can see in the diagram above, each T of the coding strand is replaced with a U in the RNA transcript. Transcription termination RNA polymerase will keep transcribing until it gets signals to stop. The process of ending transcription is called termination, and it happens once the polymerase transcribes a sequence of DNA known as a terminator. Termination in bacteria There are two major termination strategies found in bacteria: Rho-dependent and Rho- independent. In Rho-dependent termination, the RNA contains a binding site for a protein called Rho factor. Rho factor binds to this sequence and starts "climbing" up the transcript towards RNA polymerase. \ ® 1a|Page Scanned with @ camscanner Rho-dependent termination, The terminator is a region of DNA that includes the sequence that ccodes for the Rho binding site in the mRNA, as well as the actual transcription stop point (which is a sequence that causes the RNA polymerase to pause so that Rho can catch up to it). Rho binds to the Rho binding site in the mRNA and climbs up the RNA transcript, in the 5' to 3 direction, towards the transcription bubble where the polymerase is. When it catches up to the polymerase, it will cause the transcript to be released, ending transcription. ‘When it catches up with the polymerase at the transcription bubble, Rho pulls the RNA transeript and the template [NA strand apart, releasing the RNA molecule and ending transcription. Another sequence found later in the DNA, called the transcription stop point, causes RNA polymerase to pause and thus helps Rho catch up.4“44start superscript, 4, end superscript Rho-independent termination depends on specific sequences in the DNA template strand. As the RNA polymerase approaches the end of the gene being transcribed, it hits a region rich in C and G nucleotides. The RNA transcribed from this region folds back on itself, and the complementary C and G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall. 15|Page Scanned with |\CamScanner Rho-independent termination. The terminator DNA sequence encodes a region of RNA that folds back on itself to form a haispin. The hairpin is followed by a series of U nucleotides in the RNA (not pictured). The hairpin causes the polymerase to stall, and the weak base pairing between the ‘A tuicleotides of the DNA template and the U nucleotides of the RNA transcript allows the transcrip to separate from the template, ending transcription. Ina terminator, the hairpin is followed by a stretch of U nucleotides in the RNA, which match up with A nucleotides inthe template DNA. The complementary U-A region of the RNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, produces enough instability for the enzyme to fall off and liberate the new RNA transcript. TRANSLATION During translation, which isthe second major step in gene expression, the mRNA is "read" according to the genetic code, which relates the DNA sequence to the amino acid sequence in proteins Each group of three bases in mRNA constitutes a codon, and each codon specifies a particular amino acid (hence, it is a triplet code). The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein. Within all cells, the translation machinery resides within a specialized organelle called the ribosome. In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the where the ribosomes are located. On the other hand, in prokaryotic organisms, fibosomes can attach to mRNA while itis still being transcribed. In this situation, translation ‘begins at the 5 end of the mRNA while the 3° end is still attached to DNA. In all types of ces, the ribosome is composed of two subunits: the large (50S) subunit and the small (30S) subunit (S, for svedberg unit, is a measure of sedimentation velocity and, therefore, mass). Each subunit exists separately in the cytoplasm, but the two join together on the mRNA molecule. The ribosomal subunits contain proteins and specialized RNA ile ita, ribosomal RNA (tRNA) and transfer RNA (IRNA). What is a genetic code? ‘The genetic code links groups of nucleotides in an mRNA to amino acids in a protein. In translation, the sequence of nucleotides in the mRNA is “translated” into a sequence of amino acids in a polypeptide. Genetic code is therefore defined as the set of rules by which information ‘encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells. The genetic code is a set of three-letter combinations of nucleotides called codons, each of which corresponds with a specific amino acid or stop signal. Specifically, the code defines a mapping between tri-nucleotide sequences called codons and amino acids; every triplet of nucleotides in a nucleic acid sequence specifies a single amino acid. 16|Page Scanned with |\CamScanner CHARACTERISTICS OF THE GENETIC CODE 1 Triplet nature: ‘Singlet and doublet codes are not adequate to code for 20 amino acids; therefore, it was pointed out that triplet code is the minimum required. . Degeneracy ‘The code is degenerate which means thatthe same amino acid is coded by more than one base triplet. Degeneracy does not imply lack of specificity in protein synthesis. Itmerely means that a particular amino acid can be directed to its place in the peptide cchain by more than one base triplets. For example, the three amino acids arginine, alanine and leucine each have six synonymous codons. The code degeneracy is basically of 2 types: partial and complete. In partial degeneracy, the first two nucleotides are identical but the third (i.e. 3" base) nucleotide of the degenerate codon differs; for example, CUU and CUC code for leucine. degeneracy oceurs when any ofthe 4 bases can take third position and still code for the same amino acid; for example, UCU, UCC, UCA and UCG all code for serine, [Pace = i Scanned with |\CamScanner . Non-overtapping 3 ‘The genetic code is nonoverlapping, i. the adjacent codons do not overlap. + Annonoverlapping code means thatthe same letter is not used for two different codons. In other words, no single base can take part in the formation of more than one codon. 4. Commatess . hails sas or es Tan whit tne cone codon and the beginning of the next. + There are no intermediary nucleotides (or commas) between the codons. 8. Universality + Universality ofthe code means that the same sequences of 3 bases encode the same amino acids in all life forms from simple microorganisms to complex, multicelled organisms such as human beings. 6, Polarity + The genetic code has polarity, that is, the code is always readin a fixed direction, ie, in the 5 — 3" direction. «+ Itis apparent that ifthe code is read in opposite direction (i, 3° —+ 5"), it would specify 2 differem proteins, since the codon would have reversed base sequence. STEPS INVOLVED IN TRANSLATION Translation occurs in a structure called the ribosome, which is a factory for the synthesis of proteins. The ribosoine has a small and a large subunit and is a complex molecule composed of ‘several ribosomal RNA molecules and a number of proteins. Translation of an miRNA molecule by the ribosome occurs in three stages: initiation, elongation, and termination. During initiation, the small ribosomal subunit binds to the start of the mRNA sequence. Then a transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the start codon of the mRNA sequence. The start codon in all mRNA molecules has the sequence AUG and codes for methionine. Next, the large ribosomal subunit binds to form the complete During the elongation stage, the ribosome continues to translate each codon in tum. Each corresponding amino acid is added to the growing chain and linked via a bond called a peptide bond. Elongation continues until all of the codons are read. isi Page Scanned with |\CamScanner Lastly, termination oceurs when the ribosome reaches a stop codon (UAA, UAG, and UGA). Since there are no (RNA molecules that can recognize these codons, the ribosome recognizes that ‘translation is complete. The new protein is then released, and the translation complex comes apart Ribosome mage adatoms Katona Human Genome Research latte. KEY POINTS Prokaryotic gene expression is primarily controlled at the level of transcription. Eukaryotic gene expression is controlled atthe levels of epigenetic, transcription, post transcription, translation, and post-ranslation. Prokaryotic gene expression (both transcription and translation) occurs within the ‘cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the eytoplasm. + Eukaryotic géne expression occurs in both the nucleus (transcription) and cytoplasm (ransiation).. Prokaryotie versus Eukaryotic Gene Expression To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners, ~ Prokaryotic organisms are single-celled organisms that lack a defined nucleus; therefore, their DNA floats freely within the cell cytoplasm. To synthesize a protein, the processes of transcription (DNA to RNA) and translation (RNA to protein) occur almost simultancously. When the resul protein is no longer needed, transcription stops. Thus, the regulation of transcription is the primary ‘method to control what type of protein and how much of each protein is expressed in a prokaryotic wiPage ‘Scanned with |CamScanner cell. All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level. Eukaryotic cells, in contrast, have intracellular organelles that [Link] their complexity. In ‘eukaryotic cells, the DNA is contained inside the cell's nucleus where itis transcribed into RNA. The newly-symthesized RNA is then transported out of the nucleus into the eytoplasm where ribosomes translate the RNA into protein. The processes of transcription and translation are physically separtjed by the nuclear membrane; transcription occurs only within the nucleus, and ‘oceurs only outside the nucleus within the cytoplasm. The regulation of gene expression ‘can occur at all stages of the process. Regulation may occur when the DNA is uncoiled and | loosened from nucleosomes to bind transcription factors (epigenetics), when the RNA is ‘transcribed (transcriy sional level), when the RNA is processed and exported to the cytoplasm after itis transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level). Prokaryotic vs Eukaryotic Gene Expression: Prokaryotic transcription and translation occur simultaneously in the cytoplasm, and regulation occurs atthe transcriptional level. Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, and during protein translation, which takes place in the cytoplasm. Further regulation may occur through post-translational modifications of proteins. Post Transcriptional Modifications ‘The primary transcript formed undergoes modification in eukaryotes, namely splicing, tiling and capping. In this way, post-transeriptional processing helps increase the efficiency of protein synthesis by allowing only specific protein- coding RNA to go on tobe translated. A modification also takes place at the opposite end of the RNA transcript. ol Pace Scanned with |\CamScanner CAPPING: the $end of primary transcript is attached with methylguonosine with the help of ‘enzyme guanosy! transferase. The cap protects the S* end of the primary RNA transcript from attack by ribonuclease. [Link]-the inirons (regions of RNA that do not code for proteins) are cut apart from exons (regions of RNA that do code for proteins) and the exons are again joined together. these process tus catalysed by spliceosome assembled from proteins and small nuclear RNA molecules that recognize splice sites inthe primary transcrip. f [Link]-the 3' end of primary transcript is added a adenylate and not a single adenylate but ‘, ‘many adenylate also known as polyA tail. le ecard i emcyu ol stevie F polymerase (PAT AFTER these 3 steps, primary transcript is converted into m-RNA. and transferred to ribosome for translation independent mechanisms. terminated by two elements: 4 Sono ep au evans loi ‘sequence. aipase Scanned with |\CamScanner Genetic Transformation, Transduction and Conjugation ~ Genetic Information in Microbes Genetic information in bacteria and many viruses is encoded in DNA, bo some viruses we RNA Replication of the genome is essential for inheritance of genetically determined traits, Gene expression usually involves transcription of DNA into messenger RNA and translation of mRNA. into protein, Genome Organization The bacterial chromosome is a circular molecule of DNA that functions as 2 self-replicating genetic element (replicon). Extrachromosomal genetic elements such as plasmids and bacteriophages are nonessential replicons which Sen determine resistance to antimicrobial agents, productiin of virulence factors, or other functions. The chromosome replicates semiconservatively; each DNA strand serves as template for synthesis ofits complementary strand. Genetic exchanges among bacteria occur by several mechanisms. In transformation, the recipient bacterium takes up extracellular donor DNA. In transduction, donor DNA packaged in a ‘bacteriophage infects the recipient bacterium. In conjugation, the donor bacterium transfers DNA to the recipient by mating. Genetic Transformation Genetic transformation is a process that involves the introduction and expression of foreign genes in a host organism. Transformation can occur in two ways: natural transformation and artificial transformation. Natural transformation describes the uptake and incorporation of naked DNA from the cell's natural environment. Genetic transformation in Bacteria is the process of taking up free DNA from the environment and incorporating it into a recipient cell, The ability of an organism to take up DNA is called ‘competence. > Bacterial transformation is used: To make multiple copies of DNA, called DNA cloning. Tomake inrge amounts of specific human proteins, for example, human insulin, which can be used to treat people with Type I diabetes. ‘There are two major categories of gene transformation in plants on the basis of the type of involvement of a vector, ie., the Direct (or vector less/vector independent and includes both physical and chemical methods like biolistics, microinjection, electroporation, temperature- ‘mediated, electrophoresis. } > Genetic transformation is an important strategy for enhancing plant biomass (yield) or resistance in response to adverse environments factors (e.g increased resistance to diseases) 2a|Pace Scanned with |\CamScanner enetic Transduction ; Transduction is a mode of genetic transfer from one bacteria to another through a virus. There is no direct contact between the bacterial cells. Transduction in bacteria is a process by which genetic material from a bacterium is transferred and incorporated into the genome of another bacterium by a bacteriophage (bacterial virus). Bacteriophages, also known as phages, infect and replicate in bacterial cells. They transfer genetic information during their infection cycles as they se bacterial cells as hosts to make more virus copies. The term transduction is also used to refer to the process whereby a viral vector introduces foreign DNA into another cell. In this process, bacteriophages, which infect bacteria, use host cells to multiplicate and while assembling they sometimes pack the bacterial DNA with them, Later, when these viruses infect new bacterial cells, the bacterial genome that they carry may get inserted into the host genome. Transduction is commonly used in genetic engineering for inserting foreign DNA into the host ell, Genetic Conjugation ‘Conjugation is the method of transfer of genetic material from one bacteria to another placed in ‘contact. This method was proposed by Lederberg and Tatum. They discovered thatthe F-factor ‘can move between [Link] cells and proposed the concept of conjugation, Mechaniim of Bacterial Conjugation Bacterial conjugation involves the following steps: Pilus Formation ‘The donor cells (F+ cells) form a sex pilus and begin contact with an F- recipient cell. Physical Contact between Donor and Recipient Cell The plus forms a conjugation tube and enables direct contact between the donor and the recipient cells. ° Transfer of F-Plasmid “The F-factor opeis atthe origin of replication, One strand is cut at the origin of replication, and the 5? end enters the recipient cell. Synthesis of Comple:ientary Strand ‘The donor and the recipient strand both contain a single strand ofthe F-plasmid. Thus, & ~complementary strand is synthesized in both the recipient and the donor. The recipient cel now contains a copy of F plasmid and becomes a donor cell. B|Pace Scanned with |\CamScanner Gene mutation ‘A genetic mutation isa change toa gene's DNA sequence to produce something different. Itereates a permanent change to that gene's DNA sequence. A gene mutation is a change in one or more ‘genes. Some mutations can lead to genetic disorders or illnesses. Gene mutations are changes in single DNA bases, or small intragenic deletions and rearrangements. The classification of gene mutations is often used interchangeably with point ‘mutations although, genetically, they may comprise different events. Point mutations refer to single base changes, or insertions or deletions of one or a few bases, whereas gene mutations refer to base change or iitragenic additions, deletions, or rearrangements that disrupt normal gene function. These are all considered to be heritable effects because they are usually not lethal to the ‘exposed cells and are typically measured in the posttreatment generation cells. The treated cells ‘must undergo subsequent reproductive cycles for the mutations to be expressed and scored. 24a|Page Scanned with |\CamScanner types of DNA mutations are known to occur: Base substittions. Single-base substitution is known as ‘point mutation’. These are the ‘most common types of mutations that can result in silent, missense, or nonsense. © Silent mutations: Substitution of a base that occurs on the third position of the codon. Thus there is a positive possibility that an identical codon will be {generated that will code for the same amino acid sequence. Thus, no change in the {gene sequence resulting in a silent mutation. © Missense: A base substitution that leads to the generation of a gene sequence that codes for different amino acids and eventually a different polypeptide sequence. co Nonsense: A base substitution that results in the generation of a gene sequence I that truncates the translation or alteration that leads to the generation of a stop codon that eventually forms a non-functional protein is known as Nonsense mutation. 2. Deletion. As the name suggests, deletion of one or more base pairs on the DNA. This can result in a frameshift when one or more base pairs are deleted/temoved from the DNA. 3. Insertions. The addition of additional base pairs in the DNA may also be frameshifis. However, this frameshift will depend on whether or not the addition of base pairs have ‘occurred i the multiples of three base pairs: AUG GCC TGC AAA CGC TGG ormal ’ met ala cys ys arg up + ‘ aa AUG GCT TGC AMA CGC TGS met ala cys ys ang tp Aue GCC TCA AAA cco TOG ieee) a BOOMER AOA ORE wwe cco bec AAA ccc Too met ala arg ys arg uD + tape, SUS SR TSR AAT | eased met ala 6 gu asm ala 2 we accé ToG soneath SRA terion +) met ala leu gin tr lou + + AUG GCC. T6G ote SPAS met ala leu gin the wp 2s | Pace Scanned with |\CamScanner Mutagenic agent ‘A mutagen is a chemical or physical agent capable of inducing changes in DNA called mutations. Examples of mutagens include tobacco products, radioactive substances, x-rays, ultraviolet radiation and a wide variety of chemicals. Exposure to a mutagen can produce DNA mutations ‘that cause or contribute to certain diseases. ‘Any agent causing mutation is called mutagen. Mutagens can be physical mutagens, chemical ‘mutagens, or biological mutagens. The ability of a substance to induce the alterations in the base pairs of DNA or mutation is known as mutagenicity [ ‘Some examples of physical mutagens include UV or gamma radiation while Chemical mutagens include Elements such as arsenic, nickel and chromium. Some organic compounds like benzene and alkylating agents and azides represent potential chemical mutagens. Biological mutagens ‘often consist of: viruses and different bacterial species capable of initiating changes in an individual's DNA. g Site-directed mutagenesis (SDM) SDM sometimes referred to as site-specific or directed mutagenesis, is a method of altering the nucleotide sequence of a gene at a specified location, which stands in contrast to general ‘mutagenesis that employs mutagenic compounds or high energy radiation to randomly alter DNA. Site-directed mutagenesis is an invaluable tool to modify genes and study the structural and functional properties of a protein, based on the structure, function, catalytic mechanism, and catalytic residues of enzymes. Site-directed mutagenesis includes single and combinational ‘mutations. SDM is a highly versatile technology that can be used to probe gene function of an isolated gene product in the context of a complex biological system, and it can be used to engineer proteins and even entire organisms for new functions or capabilities. “These changes result in altered or improved properties of enzymes encoded by the target gene. For ‘SDM, the structural and function knowledge of an enzyme isa prerequisite to design the mutations. 26| Page Scanned with |\CamScanner SDM technique requires (1) A DNA temp ate with a target gene (2) Knowledge of the target gene's nucleotide sequence, and (3) a short primer (commonly 20 to 30 base pairs) complementary to the target sequence that is ‘modified to contain a mismatched nucleotide (typically | to 3 base pairs that will couse an amino ‘acid change). ‘The general procedure for SDM is the following: + Step 1: Separation of the two strands of template DNA. This ean be accomplished by heat ‘or an alkali solution. + Step 2: Addition of the modified DNA primer. Once the DNA primer anneals with a single strand, a DNA polymerase replicates the strand. + Step 3: The second round of replication yields a mutant DNA strand that ean be used to synthesize a modified protein. Techniques for Yerforming SDM PCR and non-PCR techniques are in vitro methods for performing SDM. In vitro synthesis has four essential components: DNA template, modified primers, deoxyribonucleic nucleoside triphosphates (NTPs [ie., dATP, ACTP, dGTP, dTTP), and DNA polymerase (thermostable or thermolabile). The polymerase chain reaction technique utilizes thermostable DNA polymerases (€.g., Taq, Pfu, and Vent), at least two different primers, and multiple heating and cooling cycles (average 30 cycles). Each cycle has three phases: denaturation (approximately 95 degrees Celsius), annealing (approximately 55 degrees Celsius), and extension (approximately 72 degrees Celsius), The denaturation phase is when the template DNA molecule separates into two single-stranded ‘molecules. The annealing phase is when the modified primer base pairs atthe sequence of interest. Lastly, the extension phase is when the annealed primers are extended according to the template strand. The advantage of PCR is that it works more favorably with different DNA templates (ie., single or double-stranded DNA and GC-rich regions) and produces millions of copies of a target ‘gene. The disadvantage is there is less sequence fidelity (ie., thermostable polymerases are more ‘error-prone in comparison to thermolabile polymerases). The non-polymerase chain reaction technique utilizes thermolabile DNA polymerases, one primer, and constant reaction temperature (eg., 37 degrees Celsius), In this strategy, the DNA template is denatured with an alkali solution or heat. The temptateis annealed with the modified primer and DNA synthesis occurs at 37 degrees Celsius. Though single or double-stranded DNA templates can be used, more favorable outcomes ‘occur if a single-stranded DNA template is used (this increases the suecess of annealing). This process produces a hybrid DNA molecule (i., one template strand anid one newly synthesized W[Page Scanned with |\CamScanner mutant strand), which can be transformed or infected into E. coli. Once in [Link], the mutant DNA and wildtype DNA can be segregated. Non-PCR has greater sequence fidelity than the PCR method; however. there is only one DNA strand generated (rather than millions) Structure of eukaryotic genome The genomes of mot eukaryotes are larger and more complex than those of prokaryotes. The structure of eukaryotic genes. Most eukaryotic genes contain segments of coding sequences (exons) interrupted by noncoding sequences (introns). Both exons and introns are transcribed to yield a long primary RNA transcript. The introns are then removed. ‘The entire gene is transcribed to yield a long RNA molecule and the introns are then removed by splicing, so only exons are included in the mRNA. Although most introns have no known function, they account for a substantial fraction of DNA in the genomes of higher eukaryotes. Ct = eer eT ow | ssn ee | "Cis a S| tne sews SCS ow Genes that are expressed usually have introns that interrupt the coding sequences. A typical cukaryotic gene, therefore, consists of a set of sequences that appear in mature mRNA (called exons) interrupted by introns. ‘The regions between genes are likewise not expressed, but may help with chromatin assembly, contain promoters, and so forth. wl Pace Scanned with |\CamScanner Gene mapping Gene may tha dry eae Proce oflosating genes win a genome, The regan of genome Therefore, itis ofen apron oer a See ely of ret inte wo scien. locate each gene in a genome. Gene mappir genes. The ae jee techniques used to identify a gene's location and distance between Inaphlng tween various sites inside a gene can also be described through gene Nowadays, i oie mapping proceduré typically involves genome sequencing and analysis of the Ine Sequence using digital methods that would let ws spot desired genes, Therefore, most ‘Gene mapping projects begin with genome sequencing. ‘Types of Gene Mapping Genetic-linkage ‘maps and physical maps are the two main categories of “Maps” used in gene ‘mapping. Both maps consist of genetic markers and gene loci. While physical maps involve actual physical distances, often measured in number of base pairs, distances of genetic maps are based on ‘genetic linkage information. There are many gene mapping methods, including comparative, physical, and genetic-linkage mapping, However, physical, and geneticclinkage mapping are more common. Genetic-linkage Mapping ‘ Genetic-linkage maps show the location ofeach gene on a chromosome and their relative distances from one another. Initially, these maps were created by tracing the inheritance of several features, like eye colour and hair colour. Blood, saliva, or tissue samples from both affected and unaffected family members are used to begin a genetic map. Saliva is the mos often used sample in genetic mapping, particularly personal genetic studies. Genetic mapping is made possible by crossing over — a regular biological occurrence during meiosis (cell division that produces sperm and egg cells). Chromosomes Line up in pairs in the ‘centre ofa cell during the first phase of meiosis, where they often “stick” to one another and exchange similar fragments of themselves, also known as crossing over. 2g|Page ‘Scanned with Genetic mapping makes it possible to determine which gene is present on each chromosome and where itis located within that specific chromosome, Based on the distance between two genes, mapping can also determine which gene is more likely to undergo recombination. Physical Mappilig Physical maps always provide the actual DNA base pair distances between landmarks. It is one ‘gene mapping [Link] that has a high degree of accuracy in determining the sequence of DNA base pairs. . ‘A physical map provides the nucleotide numbers and the precise physical distance between genetic markers. Radiation hybrid mapping, sequence mapping, and cytogenetic mapping are the techniques used to produce a physical map. Physical mapping assembles larger DNA sections using DNA markers and DNA fragments. Researchers can identify the locations of the DNA bases from the overlapping sections of the fragments. Various physical mapping methods are available to study genomes of multiple sizes and obtain different degrees of precision. Physical mapping is a common approach used in genome sequencing to acquire an entire genome sequence and determine whether there is any correlation ‘between the speci‘ic DNA sequence and phenotypic features, Genetic Mapping Techniques Recombination evenis are used in genetic mapping techniques to measure the distance between ‘genetic markers, + Random Fragment Length Polymorphism, or RFLP, measures the differences in homologous DNA sequences to calculate the distance between two markers. ‘+ Currently, gene mapping analyses targeting single gene disorders use short tandem repeat polymorphisms (STRP).- + SNP (Single Nucleotide Polymorphism) is used in genome-wide association and linkage analysis genetic research. Linkage analysis is studied using the inheritance of characteristic and genetic signatures like SNPs and microsatellites. ‘+ Genome-Wide Association (GWA) studies the connections between traits and markers like SNPS and microsatellites by treating the population as a single family. The method is used to map the gene functions of common disorders. i . 30|Page Scanned with |\CamScanner solation of DNA (gene) Itis now possible to isolate a specific region of a genome, to produce a virtually unlimited number of copies of it, and to determine the sequence of its nucleotides overnight. ‘As the structure of DNA is fundamentally the same in all living organisms, theoretically any segment of DNA from any organism can be introduced in any host resulting in recombinant DNA. But the trick lies fa picking the right DNA fragment and inserting at the right place in a vector to suecessfully produce a recombinant plasmid ‘The polymerase chain reaction (PCR) PCR is now one of the most commonly used assays for obtaining a particular segment of DNA. It is rapid and extremely sensitive. By amplifying a designated segment of DNA, it provides a means to isolate that particular DNA segment or gene. This method requires knowledge of the nucleotide sequence a the ens ofthe repon that you wish o amplify How PCR Works Basically, PCR is DNA replication on a grander scale, The polymerase chain reaction relies on the use of several essential chemical ingredients, including the following: 1 + ADNA polymerase ‘+ A small amount of DNA to serve as the initial template + The four deoxyribonucleotides to serve as the substrates for the DNA polymerase and the raw ingrédients of the new DNA molecules. There are four types of dNTP, or deoxynucleotide triphosphate, with each using a different DNA base: adenine (dATP), cytosine (ACTP), guanine (4GTP), and thymine (dTTP). Using dNTP during the extension phase provid.s single bases ready to go into DNA and double it, like building blocks. +A few necessary ions and salts + A pair of primers with exposed 3-OH groups that will bind to the particular sequence of interest in the DNA template Complementary DNA (¢DNA) synthesis ‘DNA is synthesized from messenger RNA . cDNA is not genomic DNA, because the transcript ‘of genomic RNA has been processed (i... it lacks promoters and introns). The enzyme reverse transcriptase is used to synthesize double-stranded DNA that is a complimentary copy of the mRNA What techniques are used to produce eDNA? + cDNA is produced in two basic steps: (a) first, mRNA is isolated from other cellular RNA using an elution column. (b) Second, the enzyme reverse transcriptase synthesizes strands 01" INA using the mRNA molecules as templates. BPage Scanned with |\CamScanner f/ sesirietee enzymes ‘ot restriction endonucleases, are enzymes made by bacteria. These enzymes protect bacteria a crt foreign DNA molecules that eat tas their cells by, for ‘example, an invading bacteriophage, Each restriction enzyme recognizes a specific sequence, usually of four or six nucleotides in the DNA. These sequences, when they occur in the ‘bacterium's own DNA, are chemically modified by methylation, so that they are not recognized and degraded. Where these sequences occur in foreign DNA, they are cut by the restriction enzyme. Palindrome In molecular biology, the term palindrome means that the sequence of the recognition site when read in the 5* to 3* direction forthe top strand is exactly the same as that of the bottom strand. Consider the sequence recognized by the restriction enzyme known as Hind III (pronounced hin- dee-three). Ibis AG-CT-T-S -C-G-A-A-S* 3 ‘On the top strand. the recognition sequence is S* AAGCIT3° Recombinant DNA and its application, Recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus f all genetics is the gene, the fundamental goal of laboratory geneticists isto isolate, characterize, and manipulate genes. ! Recombinant DNA technology is playing a vital role in improving health conditions by developing ‘new vaccines and pharmaceuticals. Synthesis of synthetic human insulin and erythropoietin by genetically modified bacteria DNA cloning is tye process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for 4 medically important human protein) is first inserted into a circular piece of DNA called a 32] Page i Scanned with |\CamScanner ‘plasmid. ‘The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. Diagram showing the construction of a recombinant DNA molecule. Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains. ‘What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids, In other cases, the piece ‘of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the hurian insulin gene is expressed in £. coli bacteria to make insulin used by diabetics. ‘This recombinant insulin, or insulin made by combining DNA from multiple sources (humans and bacteria, is vow the primary treatment used by,type | diabetics. Steps of DNA cloning DNA cloning is used for many purposes. As an example, let's see how DNA cloning can be used. to synthesize a protein (such as human insulin) in bacteria. The basic steps are: 1. Cut open the plasmid and “paste” in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA). : 2 Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid. 3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it. Lets take a closer look at each step. ; 1. Cutting and pasting DNA How can pieces of DNA from different sources be joined together? A common method uses two types of enzymes: restriction enzymes and DNA ligase. 33) Page Scanned with |\CamScanner , * , restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and ‘euts DNA into two pieces at of near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone. ‘Our goal in cloning is to insert a target gene (¢.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest: + The plasmid, which has a single cut site + The target gene fragment, which has a eut site near each end ‘Then, we combiiie the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene. Palindrome In molecular biology, the term palindrome means that the sequence of the recognition site when read in the 5* to 3° direction for the top strand is exactly the same as that of the bottom strand. ‘Consider the sequence recognized by the restriction enzyme known as Hind III (pronounced hin- dee-three). Ibis 3! -A-A-G-C-T-1-3" 3) -TeT-C-G-A-ASS* 34|Page Scanned with |\CamScanner ; ,, nthe tp strand, the recognition sequence is 3° AAGCTT 3" ae Diagram depicting restriction digestion and ligation in a simplified schematic. 2. Bacterial transformation and selection Plasmids and other DNA can be introduced into bacteria, such as the harmless E col! used in labs, jin a process called transformation. During transformation, specially prepared bacterial cells are siven a shock (such as high temperature) that encourages them to take up foreign DNA. Lgehon, Serre bactena, Fokts Silas a pistrad Cy e Heat ghock ;, = L Bacteria Other lbocteria. dow: A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a sp-cific antibiotic, Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die,' while bacteria ‘carrying a plasmid can live and reproduce. Each surviving bacterium will give rise toa small, dot- like group, or colony, of identical bacteria that all carry the same plasmid, ' 351P ace Scanned with |\CamScanner LY, Boderia without o poswmid die. Sven Left panel: Diagram of plasmid, showing that it contains an antibiotic resistance gene. Right panel: all the bacteria from the transformation are placed on an antibiotic plate. Bacteria without a plasmid will die due to the antibiotic. Protein Expression Many hosts used for the production of recombinant protein are available, but the preferred choice is Escherichia «vli because it is easy to culture, has a very short life cycle and is simply ‘manipulated [Link] due to its well-known genetics. E. coli was the first host used to produce a recombinant DNA (rDNA) biopharmaceutical, enabling the approval of Eli Lilly’s DNA human insulin in 1982, [Link] host is frequently used to produce therapeutic protein to minimize the cost of production. — PD nhittin = a = 7 Hybridoma Technology/monoclonal antibody technology Hybridoma technology is one of the most common methods used to produce monoclonal aniibodies. Moneclonal antibodies ae useful in diagnostic, imaging, and therapeutic purposes and have a very high ‘linical significance. 36) Page Scanned with |\CamScanner

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