Recombinant DNA and Gene Cloning

• Biotechnology involves manipulating organisms or their components to produce useful products.
• This practice dates back to ancient times, such as using yeast for beer and bread and selectively breeding animals.
• Biotechnology now refers to DNA technology and modern lab techniques for studying and manipulating genetic material.
• Scientists can extract genes from one organism and transfer them to another.

Recombinant DNA

• Recombinant DNA methods combine DNA from different sources to create a single molecule in a lab setting.
• Recombinant DNA technology is widely used in genetic engineering to manipulate genes for practical purposes.
• This includes genetically engineering bacteria to produce chemicals or transferring genes between organisms.

Plasmids

• Bacterial plasmids, small circular DNA molecules replicating independently, are used to manipulate genes.
• Plasmids carry few genes, are easily transferred into bacteria, and are inherited.
• Plasmids are key tools for DNA cloning, producing many identical copies of a target DNA segment for mass production.
• A plasmid isolated from E. coli bacterium

Gene Cloning Steps

• Gene cloning isolates and mass produces desired genes.
• A biologist isolates bacterial plasmid DNA and foreign DNA from various sources.
• To create recombinant DNA, both DNAs are treated with a cutting enzyme that cleaves the plasmid at a single site.
• Source DNA, much longer than the plasmid, is cut into many fragments, only one carrying gene V.
• Cut DNA from the plasmid and source are mixed; single-stranded ends of the plasmid base-pair with the target DNA fragment's ends.
• DNA ligase joins the DNA molecules by forming covalent bonds, resulting in recombinant DNA.
• The recombinant plasmid enters bacteria via transformation.
• A large clone of cells producing protein V in marketable quantities eventually grows.
• The recombinant bacterium reproduces, cloning genetically identical cells, each carrying gene V.
• This process, involving a gene-carrying DNA segment, is gene cloning.

Purposes of Gene Cloning

• The cloned gene can be used directly in further genetic engineering projects
• Recombinant bacteria mass produce medical proteins like insulin.

Restriction Enzymes

• Each restriction enzyme recognizes a specific, short DNA sequence called a restriction site.
• After binding, the enzyme cuts both DNA strands at precise restriction fragments.
• Restriction enzymes chop up foreign DNA as a bacterial defense, while the bacteria's own DNA is protected by methyl groups.
• Restriction Enzyme (EcoRI) is an enzyme from E. coli that recognizes GAATTC and cuts it, creating restriction fragments.

DNA Ligase

• DNA ligase creates covalent bonds that join the sugar-phosphate backbones of DNA strands, making the union permanent.

Recombinant DNA formation

• Recombinant DNA forms by joining restriction fragments from different sources. Sticky ends use hydrogen bonds.
• Restriction enzymes "cut and paste" DNA.

Nucleic Acid Probes

• Synthesize Complementary Strand: create a short, single DNA strand with the complementary sequence.
• Labeling the Probe: The nucleic acid probe is labeled with a radioactive isotope or fluorescent tag to make it detectable.
• Using the Probe: the nucleic acid probe identifies a specific gene or nucleotide sequence.
• The labeled probe binds to the target DNA's complementary sequence.

Function of radioactive DNA probe

• Tags the correct molecules by hydrogen-bonding to the complementary sequence in the gene of interest.
• Probe molecules bind to and label DNA only from the cells containing the gene of interest.

Applications of Nucleic Acid Probes

• Used in DNA microarrays to test the expression of multiple genes simultaneously.
• Tagged colony cells grow further to collect the gene of interest or its protein product in large amounts.
• Chemical treatment breaks cells and separates DNA strands. DNA soaks in probe solution.
• Bacterial colonies carrying the gene of interest are tagged on the filter paper for easy identification.

Genetic Information

• Genes provide instructions for making specific proteins; RNA bridges DNA and protein synthesis.
• DNA is transcribed into RNA, then translated into protein; this represents the flow of information: DNA to RNA to protein.
• Transcription and translation are linguistic terms linked to genetic information from genotype to phenotype.
• Nucleic acids and proteins have their own "languages."
• The language of nucleic acids involves the sequence of nucleotide monomers in DNA (A, T, C, G) and RNA (A, U, C, G).
• DNA's language is a linear sequence of nucleotide bases on a polynucleotide. Specific base sequences form the genes.
• RNA is synthesized using DNA as a template.
• The RNA nucleotide sequence determines the polypeptide's amino acid sequence.
• RNA carries genetic information from DNA to guide protein synthesis.
• During translation, the RNA nucleotide sequence converts to a polypeptide's amino acid sequence; the sequence is read as codons.
• Experiments confirm the flow from gene to protein is a triplet code, where the genetic instructions are written in DNA and RNA as codons.
• Codons are a set of three-nucleotide long "words" specifying the amino acids for polypeptide chains.
• A minimum of 300 nucleotides are needed to code for 100 amino acids

Genetic Code

• Molecular biologists deciphered the genetic code in the 1960s.
• Of the 64 codons, 61 code for amino acids.
• AUG codes for methionine and signals the start of a polypeptide chain.
• UAA, UGA, and UAG are stop codons, marking the end of translation.
• RNA codons have a straightforward, complementary relationship to DNA codons.
• Codons are arranged linearly in DNA and RNA, without gaps.
• There is redundancy in the genetic code example UUU and UUC both specify phenylalanine but no ambiguity, each codon only represents one specific amino acid.
• The genetic code is nearly universal across all organisms.
• This universality enables modern biotechnology to combine genes from different species and suggests the code evolved early in life.
• RNA molecule's nucleotide bases are complementary to those on the DNA strand.

Transcription

• Transcription transfers genetic information from DNA to RNA.
• Focus is on transcription in prokaryotic cells, which is simpler than in eukaryotic cells.
• During transcription, one DNA strand serves as a template for RNA; the other is unused.
• RNA polymerase moves along the gene, forming an RNA strand by base-pairing rules, with U replacing T.
• Occurs in three main stages: Initiation, Elongation, and Termination.

Initiation:

• RNA polymerase attaches to the promoter region on the DNA.
• Once attached, RNA polymerase opens the double helix and starts synthesizing RNA.
• Special DNA sequences mark the start (promoter) and end (terminator) of a gene.
• The promoter acts as a binding site for RNA polymerase and determines where transcription starts.

Elongation:

• RNA strand grows as RNA polymerase moves along the gene.
• The RNA molecule peels away from its DNA template.
• Free nucleotides form hydrogen bonds with the template DNA, following base-pairing rules (with U replacing T in RNA).

Termination:

• RNA polymerase reaches the terminator DNA sequence.
• The RNA polymerase detaches from the RNA strand and the DNA.

Eukaryotic RNA

• Messenger RNA (mRNA) encodes amino acid sequences and carries messages from DNA to translation machinery.
• In prokaryotes, transcription and translation happen in the cytoplasm.
• In eukaryotes, mRNA is transcribed in the nucleus and travels to the cytoplasm.
• Before leaving the nucleus, eukaryotic transcripts are modified with a cap (modified G nucleotide) at the 5' end and a tail (50-250 A nucleotides) at the 3' end.
• These additions facilitate mRNA export, protect it from degradation, and assist ribosomes.
• The cap and tail themselves are not translated into the protein.
• Another key modification in eukaryotes is RNA splicing, necessitated by the presence of noncoding stretches of nucleotides within the coding sequences.
• During RNA splicing: Both exons (coding regions) and introns (noncoding regions) are transcribed from DNA into RNA.
• These genes have introns, noncoding sequences spliced out of the RNA transcript to produce mRNA.
• The final mRNA molecule consists of the joined exons along with the cap and tail.
• mRNA is synthesized from a DNA template by RNA polymerase
• RNA splicing is catalyzed by a complex of proteins and small RNA molecules, and allows production of multiple polypeptides from a single gene.
• This is achieved by varying the combination of exons included in the final mRNA.
• In humans, RNA splicing enables the approximately 21,000 genes to produce significantly more polypeptides by including different exons in the mature mRNA.
• Translation converts the nucleic acid language of mRNA into the protein language of amino acids, using more complex machinery than transcription.

Transfer RNA Molecules

• Translation needs an interpreter to convert mRNA's nucleic acid language to proteins.
• Cells use transfer RNA (tRNA) to convert codons into the amino acid sequences of proteins.
• tRNA transfers amino acids from the cytoplasm to the growing polypeptide in a ribosome.
• tRNA performs two key functions: (1) picking up the correct amino acids and (2) recognizing the corresponding codons in mRNA.
• Each amino acid is joined to the correct tRNA by a specific enzyme; there are 20 different enzymes.
• These enzymes use ATP to bind the appropriate amino acid to its corresponding tRNA.
• Cells producing proteins stock the cytoplasm with all 20 kinds of amino acids.
• Anticodon region varies from one type of tRNA to another.
• The anticodon is the base triplet of a tRNA molecule that couples the tRNA to a complementary codon in the mRNA.
• It pairs with a specific codon on mRNA.
• tRNA is a key step in translating mRNA to polypeptide

Ribosomes

• Coordinate the functioning of mRNA and tRNA and catalyze polypeptide synthesis.
• A ribosome consists of two subunits: a large subunit and a small subunit.
• Each is made up of proteins and ribosomal RNA (rRNA).
• Ribosomes of bacteria and eukaryotes have similar functions but differ in size and structure; eukaryotic ribosomes are slightly larger.
• Structural differences are medically significant; antibiotics like tetracycline and streptomycin can inactivate bacterial ribosomes but not eukaryotic ribosomes.
• A fully assembled ribosome has: A binding site for mRNA on the small subunit and binding sites for tRNA (called the P site and A site) on the large subunit.
• Ribosome subunits act like a vise, holding tRNA and mRNA molecules close together.
• A ribosome holds mRNA and tRNAs together and connects amino acids from the tRNAs to the growing polypeptide chain.

Elongation

• A site-tRNA Binding- The anticodon of an incoming tRNA, which carries a specific amino acid, pairs with the complementary mRNA codon in the ribosome's A site.
• The polypeptide detaches from the tRNA in the P site and forms a new peptide bond with the amino acid on the tRNA in the A site.
• The ribosome then moves the remaining tRNA (which now holds the growing polypeptide chain) from the A site to the P site.
• Elongation continues until a stop codon (UAA, UAG, or UGA) reaches the ribosome's A site, signaling the termination stage of translation.
• The completed polypeptide is released from the last tRNA, and the ribosome disassembles into its subunits.
• Initiator tRNA binds to the start codon on mRNA
• If a mutation caused a codon in the middle of an mRNA to change from UUA to UAA translation would stop prematurely

Flow of Genetic Information

• Genes encode instructions to create RNA molecules, which are then used to produce proteins.
• These proteins control organismal structures and functions, allowing genetic information to shape traits and characteristics.
• Messenger RNA synthesis copying a DNA template
• Amino acids are linked to specific tRNAs using enzymes and ATP.
• DNA does not participate directly in translation
• In eukaryotic cells, transcription takes place in the nucleus, and the mRNA undergoes processing before moving to the cytoplasm and in prokaryotic cells, transcription occurs in the cytoplasm.
• Translation occurs in the cytoplasm in 4 steps: mRNA binds to a ribosome and tRNAs bring the correct amino acids in sequence following the mRNA codons, a polypeptide is synthesized, the polypeptide folds, and the two ribosomal subunits separate, and the tRNA and mRNA are released.

Mutations

• Sickle-cell disease is caused by a single amino acid change in a hemoglobin polypeptide due to a single nucleotide difference in the DNA.
• A nucleotide substitution involves replacing one nucleotide and its base-pairing partner with another pair of nucleotides.
• Some substitution mutations, such as changing GAA to GAG, have no effect on the protein product because both codons code for the same amino acid (Glu). This type of change is called a silent mutation.
• A missense mutation changes one amino acid to another in a protein, like changing GGC (Gly) to AGC (Ser).
• Nonsense mutations convert an amino acid codon into a stop codon, such as changing AGA (Arg) to UGA (stop), leading to a prematurely terminated, likely non-functional protein.
• Frameshift mutations occur when nucleotides are added or subtracted in a number that is not a multiple of three, altering the reading frame (triplet grouping) of the genetic message.
• A substitution that changed an amino acid codon into a stop codon would produce a prematurely terminated polypeptide.
• High-energy radiation like X-rays or UV light is a physical mutagen. Chemical mutagens, such as molecules like DNA bases, disrupt DNA replication.
• Mutations are crucial for creating genetic diversity, which is essential for evolution by natural selection.