Genetic Engineering

Bionengineering

Direct, deliberate modification of an organism’s genome

Biotechnology

Use of an organism’s biochemical and metabolic pathways for industrial production

Practical properties of DNA

• Intrinsic properties of DNA hold true even in a test tube

• DNA heated from 90^oC to 95^oC - the two strands separate, and then the nucleotides can be identified, replicated, or transcribed

• Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form

Restriction endonucleases

• Recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides

• The enzymes can be used to cleave DNA at desired sites

• Recognize and clip the DNA at palindrom base sequences

• Used in the lab to cut DNA into smaller pieces - restriction fragments

Ligase

• Rejoins phosphate-sugar bonds (sticky ends) cut by endonucleases

• Used for final splicing of genes into plasmids and chromosomes

Reverse transcriptase

• Makes a DNA copy of RNA - cDNA

• cDNA can be made from mRNA, tRNA, or rRNA

• Provides a means of synthesizing eukaryotic genes from mRNA transcripts - synthesized gene is free of introns

Gel electrophoresis

• Separates DNA fragments based on size

• DNA samples are placed on soft agar gel and subjected to an electric current

• Negative charge of molecule causes DNA to move toward positive pole

• Rate of movement is dependent on size of fragment - larger fragments move more slowly

• Fragments are stained for observation

• Useful in characterizing DNA fragments and comparing genetic similarities

Nucleic acid hybridization and probes

• Single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can unite with other RNA - hybridization

• Foundation for gene probes - short DNA fragments of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample

• Useful in detecting specific nucleotide sequences in unknown samples

  • Southern blot method - DNA fragments are separated by electrophoresis, denatured, and then incubated with DNA probes. Probes will attach to a complementary segment if present

  • Isolate fragments form a mix of fragments and find specific gene sequences

DNA sequencing

Determining the actual order and type of bases of all types of DNA

Sanger technique

• Most common sequencing technique

• Test strands are denatured to serve as a template to synthesize complementary strands

• Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide

Polymerase Chain Reaction (PCR)

• Method used to amplify DNA

• Rapidly increases the amount of DNA in a sample

• Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides

• Repetitively cycled through denaturation, priming, and extension

• Each subsequent cycle doubles the number of copies for analysis

• Essentnially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies

Recombination DNA technology

• The intentional removal of genetic material from one organism and combining it with that of a different organism

• Objective of recombinant technology is cloning which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated

• The gene is inserted into a vector (plasmid, virus) that will inset the DNA into a cloning host

• Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product

Characteristics of Cloning Vectors

• Must be capable of carrying a significant piece of donor DNA

• Must be readily accepted by the cloning host

• Plasmids - small, well characterized, easy to manipulate, and can be transferred into appropriate host cells through transformation

• Bacteriophages - have the natural ability to inject their DNA into bacterial hosts through transduction

Vector Considerations

• Origin of replication is needed so it will be replicated

• Vector must accept DNA of the desired size

• Gene which confers drug resistance to their cloning host

Construction of a recombinant gene, insertion, and genetic expression

• Prepare the isolated genes for splicing into a vector by digesting the gene and the plasmid with the same restriction endonuclease enzymes creating complementary sticky ends on both the vector and insert DNA

• The gene and plasmid are placed together, their free ends base-pair, and ligase joins them

• The gene and plasmid combination is a recombination

• The recombinant is introduced into a cloning host

• The cloning host transcribes the gene and translates the protein

• Use selective media to quickly identify recombinants

Biochemical products of recombinant DNA technology

• Enables large scale manufacturing of life-saving hormones, enzymes, vaccines

• Insulin for diabetes

• Human growth hormone for dwarfism

• Erythoropoientin for anemia

• Factor VIII for hemophilia

• HBV vaccine

Genetically Modified Organisms (GMO, transgenic)

• Recombinant microbes

  • Pseudomonas syringae - prevents ice crystals

  • Bacillus thuringienisis - encodes an insecticide

• Many enzymes, hormones, and antibodies used in drug therapy are manufactured using mammalian cell culture

  • Cell cultures can modify the proteins

• Microbes to bioremediate disturbed environments

• Oncolytic adenoviruses - host range consists of cells that produce cancer-specific proteins

Gene therapy

• Correct or repair a faulty gene in humans

• Ex vivo therapy:

  • Normal gene cloned in vectors, tissue removed form the patient

  • Naked DNA or vector is directly introduced into the patient’s tissues

DNA Technology as Genetic Medicine

• Gene silencing techniques

• Anti-sense RNA

  • Has bases complementary to the sense strand of mRNA

  • Results in a loss of translation of mRNA

• Anti-sense DNA

  • Delivered to the nucleus, binds specific mRNAs

  • Blocks reading of mRNA transcript on ribosomes

Genome Analysis

• DNA Fingerprinting - Every individual has a unique sequence of DNA

• Methods used include restriction endonucleases, electrophoresis, hybridization, and Southern blot

Types of Genome Analysis

• SNP - single nucleotide polymorphism

• Markers

  • VNTRs

  • Microsatellite polymorphisms

• DNA fingerprinting - used to:

  • Identify hereditary relationships

  • Study inheritance of patterns of diseases

  • Study human evolution

  • Identify criminals or victims of disaster

• Analysis of Mitochondrial DNA is used to trace evolutionary origins