Chapter 9: Biotechnology and DNA Technology

Biotechnology

Use of microorganisms or cells to make useful products such as foods antibiotics vitamins and enzymes.

Recombinant DNA (rDNA) Technology

Insertion or modification of genes to produce desired proteins.

Vector

Self-replicating DNA molecule that carries foreign DNA into a cell.

Clone

Population of genetically identical cells arising from one original cell.

Genetic Modification Procedure

Isolate a vector and desired gene; insert gene into vector; introduce into host cell; clone cells; harvest gene copies or proteins.

Gene Cloning

Production of many identical copies of a specific gene.

Protein Production

Use of genetically modified cells to produce useful proteins such as human growth hormone.

Selection

Choosing naturally occurring microorganisms that already have desirable traits.

Mutation

Genetic change that may give microorganisms desirable characteristics.

Mutagens

Agents that cause mutations in DNA.

Restriction Enzymes

Enzymes that cut DNA at specific recognition sequences.

Natural Function of Restriction Enzymes

Protect bacteria by cutting foreign DNA such as bacteriophage DNA.

DNA Methylation

Modification that protects bacterial DNA from being cut by its own restriction enzymes.

Blunt Ends

Straight DNA cuts that produce no single-stranded overhangs.

Sticky Ends

Staggered DNA cuts that create single-stranded overhangs capable of complementary base pairing.

DNA Ligase

Enzyme that joins DNA fragments by sealing their sugar-phosphate backbones.

Restriction Enzymes vs DNA Ligase

Restriction enzymes cut DNA while DNA ligase joins DNA fragments.

Plasmid

Small circular DNA molecule commonly used as a vector in genetic engineering.

Origin of Replication (ori)

Plasmid region that allows DNA replication.

ampR Gene

Plasmid gene that provides resistance to ampicillin.

lacZ Gene

Plasmid gene that codes for the enzyme beta-galactosidase.

Polymerase Chain Reaction (PCR)

Technique used to amplify small amounts of DNA for analysis.

Uses of PCR

Detecting pathogens and diagnosing genetic diseases.

PCR Ingredients

DNA template; primers; DNA polymerase; and nucleotides.

PCR Denaturation

Heating DNA to approximately 94°C to separate its two strands.

PCR Annealing

Cooling DNA to approximately 60°C so primers attach to complementary sequences.

PCR Extension

Heating to approximately 72°C so DNA polymerase synthesizes new DNA strands.

PCR Cycles

Repeated heating and cooling that produces increasing numbers of DNA copies.

Reverse-Transcription PCR (RT-PCR)

Technique that converts RNA into DNA using reverse transcriptase before DNA amplification.

Transformation

Process in which cells take up DNA from their surrounding environment.

Electroporation

Electrical current creates temporary pores in cell membranes to allow DNA entry.

Protoplast Fusion

Removal of bacterial cell walls allows cells to fuse and combine genetic material.

Microinjection

Direct injection of foreign DNA into a cell using a tiny needle.

Genomic Library

Collection of DNA fragments representing an organism's genome.

Complementary DNA (cDNA)

DNA made from mature mRNA using reverse transcriptase; lacks introns.

Reverse Transcriptase

Enzyme that synthesizes DNA using RNA as a template.

Exons

Gene sequences retained in mature mRNA.

Introns

Noncoding sequences removed from RNA during processing.

Importance of cDNA

Allows eukaryotic protein-coding genes to be expressed in bacteria because introns are absent.

Escherichia coli (E. coli) Advantages

Easy to grow and has a well-understood genome.

Escherichia coli (E. coli) Disadvantages

Produces endotoxins and does not readily secrete protein products.

Saccharomyces cerevisiae (Yeast)

Easily grown organism that can express eukaryotic genes.

Plant Cells in Biotechnology

Express eukaryotic genes and allow inexpensive large-scale production.

Mammalian Cells in Biotechnology

Produce proteins for medical use but are more difficult to grow.

Recombinant Human Proteins

Useful proteins such as insulin and human growth hormone produced through genetic engineering.

Subunit Vaccines

Vaccines made from specific pathogen proteins that can be produced by genetically modified yeast.

DNA Vaccines

Vaccines that introduce genes encoding pathogen antigens to stimulate an immune response.

Gene Therapy

Treatment that replaces defective or missing genes.

CRISPR

Gene-editing technology used to modify DNA at specific locations.

Bioinformatics

Computer-assisted analysis of biological information to understand gene function.

Proteomics

Study of the proteins expressed in a cell.

Southern Blotting

Technique that uses labeled DNA probes to identify specific DNA sequences.

Restriction Fragment Length Polymorphisms (RFLPs)

DNA fragments of varying lengths produced through restriction enzyme digestion.

Gel Electrophoresis

Technique that separates DNA fragments according to size.

DNA Migration in Gel Electrophoresis

Smaller DNA fragments travel farther through the gel than larger fragments.

DNA Probe

Labeled DNA sequence that binds to a complementary target DNA sequence.

Southern Blotting Steps

Cut DNA; separate fragments by electrophoresis; transfer to membrane; add labeled probes; detect matching DNA.

Genetic Testing

Screening DNA for genetic disorders or disease-associated mutations.

Blue-White Screening

Technique that identifies bacterial colonies containing recombinant plasmids using lacZ activity.

White Colonies in Blue-White Screening

Contain plasmids with foreign DNA inserted into the lacZ gene.

Blue Colonies in Blue-White Screening

Contain plasmids with functional lacZ that produce beta-galactosidase.

Colony Hybridization

Uses labeled DNA probes to identify bacterial colonies containing a desired gene.

RNA Interference (RNAi)

Process that prevents protein expression by targeting specific mRNA molecules for degradation.

Small Interfering RNA (siRNA)

Short RNA molecule that guides the destruction of complementary mRNA.

RNA-Induced Silencing Complex (RISC)

Protein complex that breaks down targeted RNA to prevent protein expression.

DNA Sequencing

Determining the nucleotide sequence of DNA by analyzing and assembling DNA fragments.

Ti Plasmid

Plasmid used to transfer foreign DNA into plant cells for genetic modification.

DNA Banding Patterns

Patterns of DNA fragments used to compare samples and identify possible sources of infection.