Introduction to Biotechnology and Recombinant DNA

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• Biotechnology involves manipulating organisms or their components to create useful products.
• It includes ancient practices like using yeast for beer and selective breeding as well as modern DNA technology.
• DNA technology involves laboratory techniques for studying and manipulating genetic material.
• Scientists can extract genes from one organism and transfer them to another using these techniques.
• Recombinant DNA is formed by combining DNA pieces from different sources in vitro.
• Recombinant DNA technology facilitates genetic engineering.
• Genetic engineering enables direct manipulation of genes for practical purposes.
• Scientists have genetically engineered bacteria to mass-produce chemicals and transferred genes between species.
• Bacterial plasmids are small, circular DNA molecules replicating separately from bacterial chromosomes.
• Plasmids carry a few genes, can easily transfer into bacteria, and are passed from one generation to the next.
• Plasmids are key tools for DNA cloning.
• DNA cloning produces many identical copies of a target DNA segment.

DNA and RNA

• DNA and RNA are nucleic acids constructed of nucleotide polymers.
• Nucleotides in DNA and RNA feature different nitrogen-containing bases allowing for vast sequence arrangements in a polynucleotide.
• DNA serves as the molecule of heredity.
• The arrangement of DNA includes a double helix, an opened strand showing individual DNA polynucleotides, and a zoomed-in view of a single nucleotide
• Polynucleotides are polymers made of nucleotide monomers bonded together.

DNA Nucleotides

• Each DNA nucleotide includes a nitrogenous base (A, C, T, or G), a sugar (deoxyribose), and a phosphate group.
• Nucleotides link through covalent bonds between the sugar of one nucleotide and the phosphate of the next, forming a sugar-phosphate backbone.
• Nitrogenous bases project from this backbone.
• The phosphate group contains a central phosphorus atom surrounded by four oxygen atoms.
• Deoxyribose has five carbon atoms, four in a ring and one extending above, and lacks one oxygen atom compared to ribose.
• Its name, deoxyribonucleic acid, refers to the sugar form ("deoxyribo"), its presence in cell nuclei ("nucleic"), and the ionized phosphate group ("acid").
• Every nucleotide has a nitrogenous base like thymine.

DNA Nitrogenous Bases

• Nitrogenous bases are basic, not acidic like phosphate groups.
• Each base possesses a single or double ring structure with nitrogen and carbon atoms, plus various attached functional groups.
• Functional groups affect molecule function, as they participate in chemical reactions.
• Functional groups determine which bases can form hydrogen bonds with each other, for example, the NH2 group on cytosine can form a hydrogen bond with the C=O group on guanine, but not with the NH2 group on adenine.
• This specificity of chemical groups ensures specific base pairing in DNA.
• DNA contains four nucleotides differing in their nitrogenous bases: pyrimidines (thymine and cytosine, single-ring) and purines (adenine and guanine, double-ring).
• Bases are often abbreviated with one-letter codes for the base itself or the nucleotide containing it.

RNA

• RNA (ribonucleic acid) differs from DNA in a few ways
• RNA´s sugar is ribose with an -OH group lacking in deoxyribose (DNA's sugar)
• RNA contains uracil (U) instead of thymine.
• RNA polynucleotide chains are otherwise identical to DNA.
• RNA's backbone is chemically similar to DNA's, with yellow phosphate groups and blue ribose sugars, with a structured sugar-phosphate backbone.

DNA vs RNA Base Pairing

• Both are nucleotide polymers with a sugar, a nitrogenous base, and a phosphate group.
• RNA contains ribose, while DNA contains deoxyribose.
• Both RNA and DNA have A, G, and C bases, but DNA has T while RNA has U.

DNA Structure Discovery

• Watson and Crick are credited with discovering DNA structure.
• After the 1952 Hershey-Chase experiment, scientists competed to determine DNA's structure.
• Researchers already understood how covalent bonds arrange nucleic acid polymers.
• Watson joined Crick at Cambridge, who used X-ray crystallography to study protein structure.
• Watson saw Rosalind Franklin's X-ray image of DNA at Wilkins's lab in King's College, London.
• Watson then deduced DNA's helical shape and evenly wide nitrogenous bases stacked like plates.
• Franklin's data also showed that sugar-phosphate backbones were on the helix's outside, with nitrogenous bases inside.
• The challenge was arranging the bases inside the double helix.
• Watson and Crick built wire model of a double helix matching Franklin's data & DNA chemistry knowledge
• Initially they thought DNA bases paired like with like (A with A, C with C) but matching the physical data required the pairing of a double ring base (purine) and a single ring base (pyrimidine)
• After trail and error they determined the chemical structure dictate specific pairings: adenine (A) with thymine (T), and guanine (G) with cytosine (C).
• These pairings are complementary and maintain the helix structure.
• Watson and Crick's pairing scheme matched physical and chemical data and confirmed Chargaff's findings
• Chargaff discovered any species' DNA had equal levels of adenine (A) and thymine (T), and guanine (G) and cytosine (C).
• This is explained by A always pairing with T and G always pairing with C on DNA's polynucleotide chains, now known as Chargaff's rules.
• The DNA double helix looks like a twisted rope ladder.
• The sides of the ladder are sugar-phosphate backbones, while the rungs are nitrogenous base pairs linked by hydrogen bonds.
• Watson and Crick published their DNA model in Nature in 1953 and received a Nobel Prize in 1962 along with Wilkins.
• Rosalind Franklin contributed significantly but died in 1958 and was ineligible for the prize.
• The discovery of the double helix and its base pairing explained molecular inheritance.
• The Watson-Crick model revolutionized understanding of genes and chromosomes by aligning with the chromosome theory of inheritance.
• It revealed that genetic information is encoded in DNA's nucleotide sequence.
• DNA's structure offered a molecular explanation for genetic inheritance.

DNA Replication

• DNA's main function is encoding and storing genetic information as the molecular basis of heredity.
• During cell division and reproduction, genes transfer from one cell to the next, and from one generation to the next.
• DNA's double helix allows effective transfer and storage, relating structure and function.
• Watson and Crick proposed that complementary base pairing allows for DNA copying.
• By using one strand of the parental DNA to determine the sequence of bases covered.
• During the cell cycle the cell copies the genes along the same rules
• Parental DNA strands separate
• Each parental DNA strand stands as template for the assembly of a complementary strand from free nucleotides in the nucleus
• Enzymes link the nucleotides forming new DNA strand
• The result is identical daughter DNA molecules
• Each daughter molecule has one old strand (from the parental molecule) and one new strand.
• This semiconservative model explains how the parental molecule is conserved in each daughter molecule.
• Requires the coordination of a number of enzymes and proteins.
• The DNA molecule must untwist to replicate.
• Synthesized strands must be made near-simultaneously.
• The process is complex and usually accurate to within only one incorrect nucleotide sequence in several billion.

DNA Replication Origins and Orientation

• DNA replication starts at specific sites known as origins of replication where proteins attach to DNA, which separates the strands of the double helix.
• Replication occurs in two directions, resulting in replication "bubbles".
• The parental DNA strands open and the daughter strands elongate from each side of the bubbles.
• Eukaryotic chromosomes contain multiple origins permitting replication to begin at a number of points, reducing the amount of time needed.
• After some time the replication bubbles blend resulting in 2 completed double-stranded daughter molecules.
• The backbone of sugar phosphates runs in different orientations in each of the segments, causing the 3' (three-prime) and 5' (five-prime) ends to form.
• These refer to the carbon atoms making up the sugar of the nucleotide.
• On each of the DNA strands the 3' carbon atom is connected to a -OH group
• The 5' carbon atom on the other end of the strand is connected to a phosphate group.
• The opposite orientation is crucial to DNA replication known as antiparallel orientation.

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Continuous and Discontinuous Synthesis

• DNA Polymerases are the enzymes responsible in linking DNA nucleotides to a growing daughter strand.
• They can only add nucleotides to the 3' end of the strand, never the 5' end.
• This restriction results in new DNA being able to grow in only the 5' to 3' direction.
• Replication begins once the parental DNA strands separate which forms a replication bubble.
• Each side of the bubble has a fork shape.
• Synthesis of one of the new daughter strands can be continuous
• This occurs when the DNA polymerase is able to work towards the forking point of the parental DNA which allows nucleotides to smoothly be added in the 5' to 3' direction.
• Synthesis can also be discontinuous as the other daughter strand must be synthesized in short segments because the DNA polymerase can only work in the 5' to 3' direction.
• Okazaki Fragments forms as the replication fork opens
• Okazaki Fragments are sections synthesized on the lagging strand during replication.
• DNA ligase links Okazaki fragments into a continuous strand
• In addition to adding nucleotides, DNA polymerases proofread the result to remove incorrectly paired nucleotides
• Both polymerase and ligase are involved in repairing DNA damaged by harmful radiation and toxic chemicals.
• DNA Replication makes sure all body cells contain the same genetic information.
• Also copies instructions for the next generation.

DNA Polymerase

• The enzyme DNA polymerase joins these nucleotides to the 3' end of strand while base-pairing free nucleotides to a parental DNA strand

Genetic Expression

• An organism possess what is called a Genotype and Phenotype
• A Genotype is its genetic makeup and its nucleotide sequence makeup that creates its DNA.
• A Phenotype refers to its physical traits.
• Genetic information is expressed as molecules and proteins which results in the determination of its physical traits (Phenotype)
• Proteins (sometimes RNAs) link Genotype to Phenotype
• Proteins that are synthesized with the information dispatched via RNA are created based on the genes instructions.
• The chain of command starts with DNA in the nucleus transitioning to RNA and eventually protein synthesis in the cytoplasm.
• These steps include Trasncription (synthesis of RNA under DNA´s direction) and translation (synthesis of protein under RNA's direction).
• English Physician Archibald Garrod proposed that genes are responsible for dictating our traits through enzymes.
• For example in alkaptonuria dark urine is an example of a disease that results in the inability to produce a particular enzyme.
• American geneticists George Beadle and Edward Tatum demonstrated the ties between genes and enzymes using the bread mold, Neurospora crassa.
• Further research proved that proteins are not limited to enzymes only
• Examples of proteins include keratin and insulin that creates polypeptide chains with each chain specified by its own gene
• Hemoglobin made up of two polypeptides and of eukaryotic genes can code of a set of polypeptides through alternative splicing
• RNA transcribed doesn't only go into translation, many complete functions and this leads to the current definition of a gene to be more complex.
• This explains RNA´s genetic translation to make protein
• RNA acts as the bridge between DNA and protein synthesis.
• RNA is transcribed to DNA that then gets translated into proteins.
• The flow of information within a cell goes in the steps of DNA to RNA to protein.
• Nucleic acid is transferred between Genotype to Phenotype through what is knows as transcription and translation.
• The language used with the acids in transferred through DNA being translated to the proteins.
• The language in the acids includes the sequence of nucleotide monomers in its DNA and RNA.
• 4 types of Nitrogenous wastes include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G).
• The base uracil U, replaces Thymine in the RNA Translation
• Gene 3 which is on a DNA molecule highlights a smaller region of a gene.
• DNA´s language has nonoverlapping three-base known as words called Codons write the genetic instructions
• Codons code in a DNA pattern which is translated by mRNA which creates a Polypeptide
• 1 amino acid = 3 nucleotides and 100 amino acids = 3 nucleotides x 100

Nucleic Acid Terms of Knowledge:

• Transcription: synthesis of RNA on a DNA template.
• Transfer RNA: a type of ribonucleic acid that functions as an interpreter in translation by conveying each specific amino acid to the appropriate codon on mRNA.
• Codons: a three nucleotide sequence in mRNA that specifies a particular amino acid or polypeptide termination signal; the basic unit of the genetic code.
• Translation: synthesis of a polypeptide using the genetic information encoded in a mRNA molecule.
• Anticodon: a sequence of three bases on a transfer RNA (tRNA) molecule that is complementary to a specific codon on messenger RNA (mRNA)
• Start Codon: is a sequence that has to start with 3 nucleotides in mRNA to signal the beginning of a protein synthesis. - the codon is AUG that is required to initiate a translation.
• Triplet Code: a set of three-nucleotide long "words" that specify the amino acids for polypeptide chains.
• RNA Polymerase: a large molecular complex that links together the growing chain of RNA nucleotides during transcription, using a DNA strand as a molecule.
• Messenger RNA: the type of ribonucleic acid that encodes genetic information from DNA and conveys it to ribosomes, where the information is translated into amino acid sequences.

RNA Codons

• RNA codons have a direct and complementary relationship with DNA codons (e.g., UUU in RNA matches AAA in DNA)
• Linear Codons are arranged with DNA and RNA
• Redundancy exits in the genetic code but no ambiguity occurs as single codon only represents 1 amino acid.
• Nearly all organisms feature a Genetic code. That has allowed scientist to combine genes with different species.
• Genetic information can be seen as shared between evolutionary parties connecting the life on earth together

Transcription

• Transcription is the transfer of information from DNA to RNA. Transcription focuses on prokaryotic cells
• Transcription is also a simpler procedure in comparison to Eukaryotic Cells.
• During RNA one strand stands a new templated molecule.
• RNA forms its new strand by adding U in replacing T.
• RNA polymerase can only begin when on a set promoter sequence eventually until the enzyme can reach a terminator sequence signaling the final end of the gene
• Transcription 3-stage process
• Initiation: polymerase attaches to the promoter region and then synthesizes the RNA. The promoter is the binding site for its polymerase that starts the signal.
• Elongation: RNA strand grows once the polymerase moves across the gene Newly formed RNA breaks away from its DNA and two separated strands. Then the new nucleotides form the hydrogen bonds with the bases and the template.
• Termination: Polymerase comes into close contact with a terminator DNA ending the genes translation. The polymerase detaches from it new strand as well the DNA
• Start and end: Sequences mark the start and end of a Terminators genes to polymerize with

Eukaryotic Cells

• mRNA encodes amino acid and carries genetic messages from cells
• Translations in pro occur in translation in the Cytoplasm
• While in EU, they are transcribed in the nucleus that has to travel through cytoplasm for synthesis of its protein
• RNA undergo several modification and has a Cap (modified G nucleotide) 5' end and a long tail (50 A) at its 3'end
• mRNA travels from the nucleus exports the ribosomes binding process
• Eukaryotes has another modification in splicing where the non coding nucleotides interrupt the sequence known as Exons

RNA Splicing

• Occurs in both Exons(Coding) and non Exons
• RNA then splits from the Nucleus and merges with exons that form a continuous coding chain.
• RNA joins the tail making this molecule the one that binds
• Translation requires an interpreter and that is the tRNA to perform this action in proteins that are kept within the cell

Transfer RNA

• To translate three chains and acids used is tRNA
• 3 representation has
• Flattened Representation: the most tRNA molecule is made of roughly 80 nucleotides and has the shape of a Cloverleaf joined by hydrogen bonds
• RNA Sequence: Consistence forms through all tRNAs where the Anticodon varies
• Modified Cases: tRNA contain special chemically modified bases and these bases are required with tRNA
• The backbone eventually holds and loops. The loops have 1 end The most important parts enable the match of acids with proteins with aid of the ATP production

Ribosomes

• cells required Ribosomes to translation
• mRNA molecules, and ATP
• Ribosomes coordinate mRNA and tRNA and synthesize polypeptides
• There are two types of ribosomes: Bacteria and Eukaryotes.
• They all feature a binding site and a fully molecule mRNA
• Proximity allows the translation process across these enzymes and will allow a further connection.

Amino Acids

• The Anticodon is an incoming tRNA.
• The polypeptide is detached after this process and bond is added the acids are then added and exits through the tunnel
• The chain is the transferred in the proteins to the Acids.
• To begin the process, the tRNA exists. All while Elgonation continue till these codons can reach protein. This creates and releases subunits in the cells.

Mutations

• Mutation: caused by inherited traits can be explained by a change in the polypeptide to a hemoglobin trait.
• Nucleotide Substitution occurs for example when A replaces G within the mRNA
• A silent type of mutation has no affect but changes a protein
• Mutagen result in harmful mutations for high energy. It may result in different ways such as radiation or chemicals.
• Mutations result in proteins that enhance the effect and result of an organisms
• The protein will have a substitution with different amino acids
• The substitution had a code for the acids to produce.
• In cases of mutations where changes are bad it is a result of no diversity and the genetic selection that occurs.
• If substitution were to occurs, and an acids was damaged this can create a short proteins that in turned can create a harmful product

Gene Cloning

• In gene cloning, a molecular biologist might identify a gene coding for a valuable product, like protein V, to manufacture on a large scale.
• The challenge is isolating the gene from a longer DNA molecule.
• Gene cloning techniques can mass-produce a desired gene.
• Vector: Bacterial plasmids (e.g., from E. coli) serves as a gene carrier.
• DNA containing gene V from another organism (foreign DNA).
• The DNA containing gene V could originate from bacterium, plant, animal, or human tissue cells.
• Restriction enzymes cut both the plasmid and the gene V source DNA.
• The enzyme cleaves the plasmid in only one place.
• Source DNA cuts into many fragments.
• Millions of plasmids and DNA fragments are treated simultaneously.
• Cut DNA from both sources mixes and base-pairs.
• DNA ligase joins the two DNA molecules via covalent bonds.
• DNA ligase, a “DNA pasting” enzyme, catalyzes covalent bonds between adjacent nucleotides, joining strands.
• The resulting plasmid is recombinant DNA.
• Recombinant plasmid mixes with bacteria where bacteria take up the plasmid DNA by transformation.
• Recombinant bacterium reproduces to form a clone of cells with a copy of gene V.
• Gene cloning produces many copies of a gene-carrying DNA segment.
• Uses include producing copies of the gene itself for further genetic engineering.
• Also to harvest the protein product of the cloned gene.
• A protein with medical uses, such as insulin, can be harvested in large quantities using recombinant bacteria.
• Restriction enzymes are bacterial enzymes that act as "molecular scissors" to cut DNA.
• Restriction enzymes recognize particular short DNA sequences, called restriction sites.
• After binding to a restriction site, a restriction enzyme cuts both DNA strands at precise points within the sequence.
• Cutting DNA with restriction enzymes yields DNA pieces called restriction fragments.
• The same restriction enzyme will always cut all copies of a particular DNA molecule at the same place.
• Once cut, restriction fragments of DNA can be pasted together by the enzyme DNA ligase.
• Single-stranded regions of a DNA fragment whose unpaired bases can hydrogen-bond to complementary single-stranded regions of another fragment are called "sticky ends".

Nucleic Acid Probes

• Nucleic acid probes are used to find a specific gene or nucleotide sequence within a mass of DNA.
• A researcher can synthesize a short, single strand of DNA with a complementary sequence to a known gene sequence, and label it.
• The label can be a radioactive isotope or a fluorescent tag.
• When a radioactive DNA probe is added to a DNA collection, it tags the correct molecules by hydrogen-bonding to the complementary sequence in the gene of interest.
• A probe can be applied to many DNA molecules to screen them for a desired gene.

Practical Applications of Nucleic Acid Probes

• Nucleic acid probes can be used in DNA microarrays to test the expression of many genes at once.
• They can also identify bacterial colonies carrying a gene of interest.
• Filter paper is pressed against bacterial colonies to pick up cells.
• A chemical treatment breaks open the cells and separates the DNA strands.
• The DNA strands are soaked in probe solution.
• Bacterial colonies carrying the gene of interest are tagged on the filter paper.
• These colonies can then be grown further, and the gene of interest, or its protein product, can be collected in large amounts.
• Radioactive DNA in the probe binds to and labels DNA only from cells containing the gene of interest, due to complementary DNA sequence.

Additional Definitions

• Recombinant DNA is a DNA molecule manipulated in a lab to carry nucleotide sequences from two sources, often different species.
• A vector in molecular biology is a DNA piece, usually a plasmid or viral genome, which moves genes from one cell to another.
• Restriction enzymes are bacterial enzymes that cut up foreign DNA at specific sequences called restriction sites, protecting bacteria against intruding DNA.
• Restriction enzymes are used in DNA technology to cut DNA molecules in reproducible ways, creating restriction fragments.
• Gene cloning is the production of multiple copies of a gene.
• A nucleic acid probe is a radioactively or fluorescently labeled single-stranded nucleic acid molecule used to find a specific gene or nucleotide sequence within a mass of DNA.
• The probe hydrogen bonds to the complementary sequence in the targeted DNA.