Introduction to Genetic Engineering

Introduction to Genetic Engineering

• Basic science: Focuses on fundamental processes, no direct product or application arises
• Example: microorganism duplication, exchange, and genetic information usage

Applied Science

• Applications derived from basic research
• Examples:
  • Genetic manipulation to select only advantageous genes in pigs
  • Using DNA to identify suspects in crimes
  • Fixing genetic mutations to treat diseases
  • Utilizing RNA regulatory molecules to permanently "fix" diseases

DNA: The Raw Material

• Intrinsic properties:
  • Helicase unwinds double helix as easily in lab as in a cell
  • DNA strands separate at temperatures just below boiling (96 °C)
  • Complementary strands re-form when cooled

Enzymes for Splicing, Dicing, and Reversing Nucleic Acids

• Restriction endonucleases:
  • Cut DNA crosswise at specific sites
  • Recognize foreign DNA
  • Break phosphodiester bonds between nucleotides in DNA
• Purpose: protect bacteria from bacteriophages or plasmids
• Recognition of palindromes (specific sequences that read the same forward and backward)
• Production of sticky ends (short, single-stranded overhangs) making easy incorporation of DNA fragments possible

Sticky Ends

• Staggered cuts, leaving tails of 4-5 bases
• Base pairs with complementary tails on other DNA fragments or plasmids

Ligase

• Necessary to seal sticky ends together
• Joins phosphate-sugar bonds cut by endonucleases
• Used to splice genes into plasmids and chromosomes

Restriction Fragments

• Pieces of DNA produced by restriction endonucleases
• Restriction Fragment Length Polymorphisms (RFLPs):
  • Differences in specific restriction endonucleases' cutting patterns create variations in fragment lengths

Reverse Transcriptase and Complementary DNA (cDNA)

• Reverse transcriptase:
  • Replicates HIV and other retroviruses
  • Converts RNA into DNA
• cDNA:
  • Allows direct comparison of DNA from different organisms at a specific site.
  • Made from messenger, transfer, ribosomal, and other forms of RNA
  • Useful in synthesizing eukaryotic genes from mRNA transcripts

What are Genes?

• Genes are located on a DNA molecule within a chromosome within a cell.

What are Introns?

• Introns are sections of a gene that are not included in the final mRNA molecule.
• They do not code for amino acids.

Exons vs. Introns

• Exons are coding sections of a gene; Introns are non-coding sections
• Exons comprise the final mRNA; Introns are removed during processing.

Analysis of DNA

• Gel electrophoresis:
  • Creates a pattern of DNA fragments for analysis
  • DNA migrates through a gel based on fragment size.
  • Positions determined by staining.
  • Useful in characterizing DNA fragments, and comparing genetic similarities.
• RFLP (Restriction Fragment Length Polymorphism):
  • Used for comparison of DNA.
  • Different lengths of restriction fragments show genetic difference between individuals.
• Nucleic Acid Hybridization:
  • Two different nucleic acids can unite at their complementary regions
• Gene Probes:
  • Short stretches of DNA to find complementary DNA sequences.
  • Carry reporter molecules (e.g., fluorescent dyes) for visualization during hybridization

Polymerase Chain Reaction (PCR)

• A molecular xerox machine, rapidly increasing DNA copies
• Highly sensitive (detects single cells)
• PCR uses similar events as normal DNA replication. specialized ingredients include primers and DNA polymerases
• PCR technique includes the use of a thermal cycler; basic steps include denaturation, priming, and extension.
• Real time PCR detects products during reaction, and PCR can be adapted to analyze RNA

Methods in Recombinant DNA Technology

• Recombinant DNA technology:
  • Deliberate removal of genetic material from one organism to combine with another.
  • Creation of identical copies of DNA
• Cloning:
  • Removes a selected gene from an organism (genetic donor).
  • Propagation of that gene in a different host organism.
• Cloning Vectors:
  • Plasmids and bacteriophages have the capacity to accept DNA fragments of desired size.
  • Plasmids usually contain a gene for drug resistance to the host cell.
• Cloning Hosts:
  • Organisms chosen for cloning should have a fast growth rate, be easily cultured, not pathogenic, well-characterized genomes, and be able to accept vectors into their cells

Genetic Treatments: Introducing DNA into the Body

• Gene therapy:
  • Normal gene inserted to correct a faulty gene.
  • Benefit: permanent cure of physical dysfunction.
• Strategies for gene therapy:
  • Insert normal gene into retrovirus or adenovirus vectors.
  • Incubate patient's tissues with modified viruses to transfect
  • Reintroduce transfected cells into patient's body.
  • Naked DNA or a viral vector inserted directly into patient's tissues
• Experimentation and clinical trials of gene testing:
  • Conducted on patients with genetic conditions;
  • Targeted at cancer, single-gene defects, and infections;
  • Early trials had significant implications
• Germline gene therapy:
  • Inserts normal genes into egg, sperm, or early embryo,
  • Creates heritable changes.

DNA Technology as Genetic Medicine

• Diseases from inappropriate protein expression: e.g., Alzheimer's, viral diseases, cancers