👩‍🔬Intro to Biotechnology Unit 4 – Genetic Engineering: Recombinant DNA Tech

Key Concepts and Terminology

• Genetic engineering involves the direct manipulation of an organism's genes using biotechnology
• Recombinant DNA refers to a DNA molecule constructed by combining DNA sequences that would not normally occur together
• Restriction enzymes recognize and cut DNA at specific nucleotide sequences enabling scientists to isolate and manipulate genes of interest
• DNA ligase catalyzes the formation of a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another
• Vectors are DNA molecules that can self-replicate and be used to deliver foreign DNA into host cells (plasmids, viruses)
• Cloning involves creating genetically identical copies of DNA fragments, cells, or entire organisms
  • Reproductive cloning generates an entire organism from a single cell
  • Therapeutic cloning produces embryonic stem cells for use in research and medicine
• Transgenic organisms contain genes from another species that have been introduced through genetic engineering techniques

Historical Background

• In 1953, James Watson and Francis Crick discovered the double helix structure of DNA laying the foundation for modern genetics
• The 1970s marked the birth of genetic engineering with the discovery of restriction enzymes and DNA ligase
• In 1973, Herbert Boyer and Stanley Cohen created the first recombinant DNA molecule by splicing antibiotic resistance genes into plasmid DNA
• The development of DNA sequencing methods in 1977 by Frederick Sanger revolutionized the field allowing scientists to read the genetic code
• Kary Mullis invented the polymerase chain reaction (PCR) in 1983 enabling the rapid amplification of specific DNA sequences
• Genetically engineered insulin produced in bacteria was approved for human use in 1982 marking the first commercial application of recombinant DNA technology
• The Human Genome Project, completed in 2003, provided a complete sequence of the human genome accelerating genetic research and personalized medicine

DNA Structure and Function

• DNA (deoxyribonucleic acid) is the hereditary material found in all living organisms
• DNA is composed of four nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
• Nucleotides are joined together by phosphodiester bonds forming a long chain with a sugar-phosphate backbone
• The double helix structure of DNA consists of two complementary strands held together by hydrogen bonds between base pairs (A-T and G-C)
• The sequence of nucleotide bases along the DNA strand encodes genetic information for the synthesis of proteins
  • Genes are specific segments of DNA that code for particular proteins
  • The genetic code is read in groups of three bases called codons each specifying a particular amino acid
• DNA replication is the process by which DNA makes an exact copy of itself during cell division ensuring genetic information is passed to daughter cells
• Mutations are changes in the DNA sequence that can alter gene function and lead to genetic variation or disease

Enzymes in Genetic Engineering

• Restriction endonucleases (restriction enzymes) are bacterial enzymes that recognize and cleave DNA at specific palindromic sequences
  • Type II restriction enzymes (EcoRI, BamHI) are most commonly used producing sticky ends with single-stranded overhangs
  • Sticky ends allow the insertion of foreign DNA fragments with complementary ends into a vector
• DNA ligase seals nicks in the sugar-phosphate backbone joining DNA fragments together to create recombinant DNA molecules
• Reverse transcriptase is an enzyme used to synthesize complementary DNA (cDNA) from an RNA template
  • cDNA is more stable than RNA and can be cloned into vectors for gene expression studies or protein production
• DNA polymerases catalyze the synthesis of new DNA strands during replication and are used in PCR to amplify specific DNA sequences
• Alkaline phosphatase removes 5' phosphate groups from DNA preventing self-ligation and increasing the efficiency of recombinant DNA construction

Recombinant DNA Techniques

• Recombinant DNA technology involves the insertion of DNA from one organism into the genome of another creating a genetically modified organism (GMO)
• The basic steps of recombinant DNA construction include:
  1. Isolation of the gene of interest using restriction enzymes or PCR amplification
  2. Insertion of the gene into a suitable cloning vector (plasmid, virus) using DNA ligase
  3. Introduction of the recombinant vector into a host cell (bacteria, yeast) for replication and expression
  4. Selection and screening of transformed host cells containing the recombinant DNA
• Gel electrophoresis separates DNA fragments based on size allowing for the isolation and purification of specific DNA sequences
• Southern blotting is a technique used to detect the presence of a specific DNA sequence in a sample using a labeled probe complementary to the target sequence
• Polymerase chain reaction (PCR) amplifies a specific DNA sequence using primers, DNA polymerase, and thermal cycling
  • PCR has revolutionized genetic research enabling the rapid cloning and analysis of genes from small amounts of DNA
• DNA sequencing determines the precise order of nucleotides in a DNA molecule providing valuable information for genetic analysis and engineering

Cloning Vectors and Host Organisms

• Cloning vectors are DNA molecules capable of replicating independently in a host cell and carrying foreign DNA inserts
• Plasmids are circular DNA molecules found in bacteria that are commonly used as cloning vectors
  • Plasmids contain an origin of replication, selectable marker genes (antibiotic resistance), and multiple cloning sites with restriction enzyme recognition sequences
  • High copy number plasmids (pUC) are used for DNA amplification while low copy number plasmids (pBR322) are used for stable gene expression
• Bacteriophages (lambda phage, M13) are viruses that infect bacteria and can be engineered to carry foreign DNA inserts
• Cosmids are hybrid plasmid-phage vectors that can accommodate larger DNA inserts (up to 45 kb) than plasmids alone
• Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are used for cloning extremely large DNA fragments (100-300 kb)
• Escherichia coli is the most common bacterial host for recombinant DNA due to its rapid growth, well-characterized genetics, and ease of genetic manipulation
• Saccharomyces cerevisiae (baker's yeast) is used as a eukaryotic host for studying gene expression and protein modifications in a more complex cellular environment

Applications in Biotechnology

• Production of recombinant proteins (insulin, growth hormone) in bacterial or yeast hosts for therapeutic use
• Genetically engineered crops with improved traits such as herbicide resistance (Roundup Ready soybeans), insect resistance (Bt corn), and enhanced nutritional content (Golden Rice)
• Transgenic animals as disease models (Oncomouse for cancer research), bioreactors for producing human proteins (ATryn antithrombin in goat milk), and organ donors (xenotransplantation)
• Gene therapy involves the introduction of functional genes into cells to replace defective or missing genes in genetic disorders (SCID, cystic fibrosis)
• DNA fingerprinting uses PCR and restriction fragment length polymorphisms (RFLPs) for forensic identification, paternity testing, and evolutionary studies
• Recombinant vaccines produced in genetically engineered organisms (hepatitis B surface antigen in yeast) offer improved safety and efficacy over traditional vaccines
• CRISPR-Cas9 is a revolutionary gene editing tool derived from bacterial immune systems that allows precise modification of DNA sequences in living cells

Ethical Considerations and Debates

• The use of genetically modified organisms (GMOs) in agriculture has raised concerns about potential ecological impacts, allergenicity, and labeling of GM foods
• Gene patenting and intellectual property rights for genetically engineered products have been controversial with arguments for incentivizing innovation versus limiting access and stifling research
• Genetic privacy and discrimination based on genetic information are major issues as genetic testing becomes more widespread
  • The Genetic Information Nondiscrimination Act (GINA) prohibits discrimination in health insurance and employment based on genetic information
• Human germline gene editing, which would introduce heritable genetic modifications, is currently prohibited due to safety and ethical concerns
  • Somatic gene editing, which affects only the treated individual, is being explored for treating genetic diseases but still raises ethical questions
• Cloning of human embryos for reproductive purposes is banned in many countries due to ethical and social objections
• The use of embryonic stem cells derived from cloned human embryos has been debated due to the destruction of embryos and the potential for commodification of human life
• Bioterrorism and the potential misuse of genetic engineering for developing biological weapons are serious security concerns requiring international oversight and regulation