Host Defenses and Innate Immunities

Genetic Engineering

• Basic knowledge is used to derive applied science or useful products.
• Bioengineering is the direct, deliberate modification of an organism's genome.
• Biotechnology utilizes an organism's biochemical and metabolic pathways for industrial production.

Tools and Techniques of Genetic Engineering

• DNA heated to 90-95°C causes the two strands to separate, allowing nucleotides to be identified, replicated, or transcribed.
• Slow cooling of DNA allows complementary nucleotides to hydrogen bond, allowing DNA to regain its double-stranded form.
• Heat denatures DNA by breaking hydrogen bonds and separating strands; cooling allows strands to rejoin at matching sites, even from different organisms.

Enzymes for Dicing, Splicing, and Reversing Nucleic Acids

• Restriction endonuclease recognizes specific DNA sequences and breaks phosphodiester bonds between adjacent nucleotides.
• Restriction endonucleases can be used to cleave DNA at desired palindromic sites.
• Restriction endonucleases are used to cut DNA into restriction fragments, which can differ in length (RFLPs).
• Action of restriction endonucleases involves recognizing and cleaving DNA at specific palindromic sequences, often producing staggered tails called sticky ends for gene splicing.
• Sticky ends join DNA from different organisms if cut by the same restriction enzyme, ensuring complementary ends.
• Ligase is necessary to rejoin phosphate-sugar bonds (sticky ends) cut by endonucleases.
• Ligase is used for the final splicing of genes into plasmids and chromosomes.
• Reverse transcriptase makes a DNA copy of RNA (cDNA) from mRNA, tRNA, or rRNA.
• Reverse transcriptase can synthesize eukaryotic genes from mRNA transcripts, which are free of introns.

Analysis of DNA

• Gel electrophoresis separates DNA fragments based on size.
• DNA samples are placed on soft agar gel and subjected to an electric current in gel electrophoresis.
• The negative charge of the DNA molecule causes it to move toward the positive pole during gel electrophoresis.
• The rate of movement in gel electrophoresis depends on fragment size, with larger fragments moving more slowly (compared to a DNA size ladder).
• Nucleic acid hybridization unites single-stranded DNA with other single-stranded DNA or RNA, or RNA with other RNA.
• Gene probes, short DNA fragments of a known sequence, base-pair with complementary DNA stretches in a sample.
• The Southern blot method separates DNA fragments by electrophoresis, denatures them, and incubates them with DNA probes. Probes attach to complementary segments if present.
• Fluorescent in situ hybridization (FISH) applies probes to intact cells and observes for presence/location of specific sequences for diagnostics or gene location on a chromosome.

Research Fields in the Study of the Genome

• Genomics is the systematic study of an organism's genes and their functions.
• Transcriptomics involves the analysis of all RNA molecules in a cell.
• Proteomics involves the study of an organism's complement of proteins and functions mediated by the proteins.
• Metagenomics is the study of all the genomes in a particular ecological niche, as opposed to individual genomes from single species.
• Metabolomics studies the complete complement of small chemicals present in a cell at any given time.

DNA Sizing, Sequencing, and Synthesizing

• DNA sequencing determines the actual order and type of bases for all types of DNA.
• The most common sequencing technique is the Sanger technique.
• Sanger sequencing denatures test strands to serve as a template, then complementary strands synthesize.
• Sanger technique separates fragments into tubes containing primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide.
• Polymerase Chain Reaction (PCR) amplifies DNA, rapidly increasing the amount of DNA in a sample.
• PCR adds primers of known sequence to indicate where amplification begins, with heat-tolerant DNA polymerase and nucleotides.
• PCR repetitively cycles through denaturation, priming, and extension.
• Each PCR cycle doubles the number of copies for analysis.
• PCR is essential in gene mapping, genetic defect/cancer studies, forensics, taxonomy, and evolutionary studies.

Sanger Sequencing Method

• Original DNA is sequenced with the Sanger Sequencing Method.
• DNA denatures to produce a single template strand for the Sanger Sequencing Method.
• The Sanger Sequencing Method labels a strand with a specific primer molecule.
• A reaction tube in the Sanger Sequencing Method contains DNA polymerase, primed single strands four all regular nucleotides
• Sanger: Small amount of the four fluorescently labeled dideoxy (dd) nucleotides that provide a visible tracer of the locations of terminal bases added by the polymerase.
• Each new strand terminates during replication at the point of addition of a dd nucleotide.

Polymerase Chain Reaction (PCR)

• DNA is amplified in cycle 1, denatured, primed, and replicated by a polymerase that can function at high temperatures.
• Resulting strands are then used for a second cycle of denaturation, priming, and synthesis.
• DNA Sample is made, then begins Denaturation with seperation of strands in cycle 1
• An Oligonucleotide primers attach at ends of strands to promote replication of amplicons during priming Heat-stable DNA polymerase synthesizes complementary strand for extension.

Recombinant DNA Technology

• Recombinant DNA technology is the intentional removal of genetic material from one organism and combining it with that of a different organism.
• The objective of recombinant technology is cloning, which requires donor gene selection, excision by restriction endonucleases, and isolation.
• The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host.
• The cloning host is usually bacterium or yeast, which can replicate the gene and translate it into a protein product.

Characteristics of Cloning Vectors

• Vectors must carry a significant piece of donor DNA and which must be readily accepted by the cloning host.
• Plasmids are small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation.
• Bacteriophages naturally inject their DNA into bacterial hosts through transduction.
• Vectors need an origin of replication and must accept DNA of the desired size and confers drug resistance to their cloning host.

Desirable Features in a Cloning Host

• A desirable Microbial Cloning Host has a rapid turnover, fast growth rate.
• A microbial cloning host can be grown in large quantities using ordinary culture methods.
• For microbial cloning a genome must be well delineated (mapped) is capable of foreign genes to multiply.
• Nonpathogenic cloning hosts should maintain foreign genes through multiple generations.
• The cloning host should secrete a high yield of proteins from expressed foreign genes.

Construction of a Recombinant - The recombinant plasmid is introduced

• In transformation the competent cloning host bacteria, which are grown on media that select only clones that are antibiotic resistant and carry the donor gene.
• Recombinant isolates are grown in culture to increase numbers and will now contain a replicated plasmid carrying the donor gene.
• Recombinant cells are induced to transcribe and translate the donor gene
• Protein products from the culture and purified from this yields. Selective media can be used to quickly identify recombinants.

Protein Products of Recombinant DNA Technology

• Recombinant DNA Technology enables the large-scale manufacturing of medications (hormones, enzymes, vaccines).
• Recombinant DNA produces Insulin for diabetes and Human growth hormone for dwarfism/Erythropoietin for anemia
• Proteins that can be generated by recombinant DNA Factor VIII for hemophilia/HBV vaccine
• Immune Treatments: Interferons peptides treat some types of multiple sclerosis, and viral infections such as hepatitis and genital warts.
• Interleukins act to: Regulate types of white blood cells; uses on Cancer treatment for bone marrow.

The following Hormones with use of Treatment:

• Erythropoietin (EPO), stimulates bone marrow treatment for anemia; can be replaced
• Tissue plasminogen activator/Hemoglobin/ Human growth.

Various Enzyme Treatments

• Cystic Fibrosis lung secretions rDNase, thick lung
• Anitrypsin acts to benefit emphysema
• PEG-SOD after severe trauma
• Adding Lipases helps laundry detergents.

Miscellaneous Vaccine products:

• Bovines, apolipoprotein and spider silk.

Gmos: the New Recombinant World

• Recombinant organisms are introduced foreign genes for genetically modified organisms
• Recombinant microbes: modified bacteria and viruses Multicellular/transgenic plants bacteria that used to disturbed ecosystem
• Recombinant bacteria are used to disturb ecosystem - bacteria that encode pesticides.

Plants and Plamsmids

• Plant Techniques used to tumors a plant or inject plasmids with disease resistance.
• To add recombinant, the plasmids are taken up by cells, which multiply/copy the foreign gene.
• Bacterium fuses with T1 that carries the herbicide gene into the T-dna to be expressed in a seed will be part of plant's genome.

Transgenic Animal Types

• Animal strains now exist for research and disease . Mice. Pigs. Sheep. Cattle.
• Used for the genetic models study disease as well as to treat diseases and provide medicine and hormones.

Genomic DNA Profiling

Every organism has a unique sequence of DNA but vary to extent of closeness For these reasons some Techniques are :

• PCR (Increase number of gene copies): Amplification increases by copies
• Electrophoreses (Seperate DNA fragments )
• Hybridizing probes ( Primitives which link to certain sequences)

Types of genomic DNA tests

• Short tandem repeats - (STRs). MtDNAs
• Or mitotyping. Single nucleotide polymorphisms -(SNPs)

Short Trandem Profiling DNA ( STR) Testing

• Has sample and then you ampify to find large numbers of fragments DNA
• A profile to tagged DNA that are specific to human genomes is created.
• Laser detections to scan and code is computer analyzed show to what type of material is D. A profile is compared then to location and correlated the number.

DNA profiling applications consists identifying

• Identify missing forensics
• Missing diseases
• Tracing parentages

Mitochondrion-based testing

• Known as DNA which the DNA and contains base pairs with a number genes - highly conservative and consistent over a period of time. • With variations to identify the levels species even certain • Or ancient due will be useful compared Genbank or bar code Genomic.

SNP

• The polymorphs occur by random through out the genes and DNA. • Valuable for tests medicine • Known to affect genetics agents of drugs cancer and diabetes .

Microarrays Analysis

• It checks for the amount genes within each cell as well to help and devise how to treat genetics types in medical disorders. </existing_notes>