Biotechnology and DNA Technology Chapter 9

Biotechnology

Biotechnology is the use of microorganisms, cells, or cell components to make a product.

Examples of biotechnology products

Examples include foods, antibiotics, vitamins, and enzymes.

Recombinant DNA (rDNA) technology

Recombinant DNA technology involves the insertion or modification of genes to produce desired proteins.

Cell clone containing rDNA

A cell clone containing rDNA can be used to harvest many copies of the gene of interest and to express (transcribe and translate) the gene of interest to produce the protein product.

Example of recombinant DNA technology

An example is the insertion of the gene for human growth hormone (hGH) into E. coli bacteria.

Selection in biotechnology

Selection involves selecting for a naturally occurring microbe that produces a desired product.

Mutation in biotechnology

Mutation refers to the use of mutagens to cause mutations that may result in a microbe with a desirable trait.

Site-directed mutagenesis

Site-directed mutagenesis is a technique that involves making a targeted and specific change in a gene.

Restriction enzymes

Restriction enzymes are enzymes produced by many bacteria that cut specific sequences of DNA.

Function of RM systems

RM systems involve the enzymatic cleavage of foreign DNA (restriction) and the modification of self DNA to prevent autodigestion, often via methylation of cytosines.

Use of restriction enzymes in genetic engineering

Restriction enzymes have become a powerful tool for genetic engineering due to their ability to cleave DNA at specific sequences.

Restriction endonucleases

Restriction endonucleases are enzymes that bind to specific DNA sequences and cleave the DNA, typically at or near the binding site.

Types of cuts by restriction enzymes

Restriction enzymes can create blunt ends or staggered cuts known as sticky ends.

Preparation of DNA fragments and vectors

The DNA to be inserted and the DNA of the vector are cut with the same restriction enzyme, and then they are joined by DNA ligase.

Role of vectors in biotechnology

Vectors carry and transfer DNA in biotechnology.

Key requirement for vectors

Vectors must be able to self-replicate.

Examples of vectors

Plasmids and viruses can be used as vectors.

Benefit of circular form of a vector

The circular form of a vector protects it from destruction in the recipient cell.

Selective markers in vectors

Selective markers, such as metabolic genes, resistance genes, or fluorescent markers, are included in vectors to aid in tracking.

Shuttle vectors

Shuttle vectors can exist in several different species and are used to move cloned sequences among various organisms.

Polymerase Chain Reaction (PCR)

PCR is a method to make many copies of a specific nucleic acid sample rapidly, allowing for the amplification of a very small sample of nucleic acid.

DNA duplication in PCR

DNA is duplicated by multiple rounds of in vitro polymerization.

Importance of heat-stable polymerases in PCR

Heat-stable polymerases are important because they can survive successive heating and cooling cycles.

Heat-stable polymerases

Polymerases that can survive successive heating and cooling cycles, unlike most polymerases that would be destabilized by heating.

Examples of heat-stable polymerases

Polymerases from the bacteria Thermus aquaticus (Taq) and the archaean Pyrococcus furiosus (Pfu).

Reagents required for PCR

Template DNA, Deoxyribonucleotide Tri Phosphates (dNTPs), primers, thermostable polymerase (such as Taq or Pfu), and a buffer to regulate pH and contain necessary ions.

Role of primers in PCR

Primers are designed to hybridize with the template DNA and must be unique sequences complementary to DNA in or near the target gene, with their 3' ends pointing towards the gene target.

Main steps in the PCR process

Denaturing, annealing, and extension/elongation, typically repeated for 30 cycles.

Denaturing step of PCR

The solution is heated to ~95°C for ~30 seconds to melt the DNA, breaking hydrogen bonds and separating the strands.

Annealing step of PCR

The solution is cooled to 50-65°C for ~30 seconds to allow the primers to anneal to the template single-stranded DNA.

Extension/elongation step of PCR

The solution is heated to ~75°C (for Taq polymerase), and the thermostable polymerase synthesizes the new DNA strand.

Thermocycler

A PCR machine used to automate the repeated heating and cooling cycles required for the PCR process.

Transformation

The process by which cells take up DNA from the surrounding environment.

Competent bacterial cells

Bacterial cells that have been treated to be able to take up external DNA.

Electroporation

A technique that uses an electrical current to form pores in cell membranes, facilitating the uptake of DNA.

Protoplast fusion

The process of removing the cell walls from two bacteria, allowing them to fuse and potentially recombine their DNA.

Gene gun

A device that fires heavy metal balls coated with DNA molecules into cells to facilitate DNA insertion.

Microinjection

A technique that uses a tiny micropipette to inject DNA directly into cells.

Genomic libraries

Collections of clones containing different DNA fragments, where an organism's DNA is digested and spliced into vectors introduced into bacteria.

Reverse-transcription PCR

A technique that uses mRNA as a template for a DNA-dependent RNA polymerase.

Complementary DNA (cDNA)

DNA made from mRNA by reverse transcriptase, used to identify the expression profile of a cell or group of cells.

Use of cDNA for eukaryotic genes

cDNA is used because eukaryotic DNA contains introns that do not code for protein, while mRNA has the introns removed.

mRNA

mRNA has the introns removed, coding only for the protein product, making cDNA a useful tool for studying gene expression.

Clone selection

Clone selection is used to identify and select cells that have successfully taken up and integrated the provided DNA, as not all cells will do so.

Blue-white screening

Blue-white screening uses a plasmid vector containing an ampicillin resistance gene (ampRR) and a β-galactosidase gene (lacZ). The cut sites for gene insertion are within the lacZ gene. Insertion of the target gene disrupts lacZ, preventing its expression. Bacteria are grown on media containing X-gal and ampicillin. Cells with no plasmid do not grow due to ampicillin. Cells with the unaltered plasmid grow and turn blue, while cells with the modified plasmid grow but remain white.

Colony hybridization

Colony hybridization is used to identify cells that carry a specific cloned gene. DNA probes, which are short segments of single-stranded DNA complementary to the desired gene, are labeled with an enzyme or fluorescent dye for visualization.

E. coli

E. coli is frequently used because it is easily grown and its genomics are well understood. However, it has disadvantages such as producing endotoxins and not secreting protein products, making harvesting the product inefficient and expensive.

Saccharomyces cerevisiae

Yeast is easily grown, has a larger genome than bacteria, expresses eukaryotic genes easily, and may continuously secrete the desired product.

Plants and plant cells

Plants and plant cells express eukaryotic genes easily, are easily grown, and can be cultivated on a large scale at low cost.

Mammalian cells

Mammalian cells are harder to grow but express eukaryotic genes easily and can produce products for medical use.

Plant breeding and biotechnology

Plant breeding has been revolutionized by the ability to grow cells in culture and alter them using recombinant DNA (rDNA) techniques.

Methods to introduce rDNA into plant cells

Methods include protoplast fusion, gene guns, and the use of the Ti plasmid from Agrobacterium tumefaciens.

Ti plasmid

The Ti plasmid occurs in Agrobacterium tumefaciens and can integrate into the plant genome, causing a tumor-like growth. It can be used to introduce rDNA into a plant.

Applications of the Ti plasmid vector

Applications include Bt toxin (insecticide) production in plant tissues, herbicide (glyphosate) resistance, suppression of genes using antisense DNA, improved nutrition, and production of human proteins.

Steps in using the Ti plasmid for plant recombination

The T-DNA is cut from the plasmid, foreign DNA is inserted into the T-DNA,

The plasmid is replaced into the bacterium, and the bacterium inserts the T-DNA carrying the foreign gene into the plant cell chromosome.

A plant is then generated from a cell clone carrying the foreign gene.

Safety and ethical issues in DNA technology

It is impossible to prove that genetic modifications are safe under all conditions. There must be precautions to avoid accidental release into the environment. Genetically modified crops must be safe for consumption and the environment. Human genomics raises questions about who will have access to genetic information and how it will be used.

DNA fingerprinting

DNA fingerprinting is used to identify pathogens and can potentially identify individuals involved in a crime through their microbiome. PCR microarrays and DNA chips can screen samples for multiple pathogens.

Applications of microbial forensics

Microbial forensics has been used in criminal cases such as rape, sexual abuse, and potential bioterror incidents. It requires proper evidence collection and establishing a chain of custody.