Chapter 20: Biotechnology

Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome.
Difficulties: Long DNA, genes are small portion, genes are identical to NC sequences.
Solutions: Small pieces, dissect and analyze, many copies and express them.

How are plasmids used to copy desired DNAs?
Naturally occurring small circles of DNA. Cut, rearranged, sealed with enzymes. Bacteria copy the plasmid = gene cloning.

Describe the natural function of restriction enzymes.
Made by bacteria to cut up phage DNA. Host DNA protected by methylation.

Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule.
Hundreds of restriction enzymes known, each cuts at specific sites leaving the same complementary ends. Mixing DNAs allows for the sticky ends to bind. DNA ligase seals backbones and recombinant DNA is created.

Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid.
Transform the recombinant DNA into bacteria. Identify desired clone: Allow only plasmid carrying bacteria to grow (AMP) and add fragments for an easily recognizable phenotype (GLO).

Define and distinguish between genomic libraries using plasmids, phages, and cDNA.
Plasmid: Thousands of randomly cut pieces inserted into plasmids. Referred to as “bacterial artificial chromosome”.
Phage: Thousands of pieces inserted into phages.
cDNA: Cloned copies of mRNA. Isolated mRNA reverse transcribed with reverse transcriptase. Inserted in plasmids and phages.

Describe the use of a nucleic acid probe to identify a desired clone.
A probe is a short radioactive DNA complementary to gene of interest. Blot plates to pick up DNA, heat to separate strands, add probe, overlay with X-ray film, and ID plate location of complementary clone carrying gene of interest..

Describe the role of an expression vector.
Goal is to overcome differences between prokaryotes and eukaryotes. Prokaryotic promoter next to restriction site. Inserted eukaryotic gene now under host control. Problem: introns.

Explain how eukaryotic genes are cloned to avoid the problems associated with introns.
mRNA lacks introns. mRNA –> cDNA using reverse transcriptase. cDNA inserted into vector.

Describe four advantages of using yeast cells instead of bacteria as hosts for cloning or expressing eukaryotic genes.
Easy to grow and have plasmids.
Process pre-mRNA.
Longer portions inserted into YACs.
Can be modified to perform post-translational mod.

Describe three techniques to aggressively introduce recombinant DNA into eukaryotic cells.
Electroporation- breaks membrane with electricity.
Micromanipulation- microinjection of single cells.
DNA-coated metal pellets- fired into cells with a gun.

Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure.
Advantage: Quick, specific, sensitive. Amplifies only sequence of interest.
Process: Cycle (Heat to separate strands, cool to allow primers to bind, heat-resistant polymerase adds to primers, repeat)
Limitations: Relatively small amounts of DNA, Errors amplified.

Explain how gel electrophoresis is used to analyze nucleic acids and proteins and to distinguish between two alleles of a gene.
Separates molecules by size, shape, charge.
Nucleic acid cut by specific restriction enzymes for a consistent set of pieces. Separate by electrophoresis. Each band is a specific piece of DNA.

Describe the Southern blotting procedure and explain how it can be used to detect and analyze instances of restriction fragment length polymorphism (RFLP).
RFLP: Individuals vary in one base sequence. If one DNA differs in a restriction site, it will not be cut. Results in a different pattern of bands.
Southern Blotting: Cut with restriction enzymes, run the gel. Blot.

Describe the dideoxy chain termination method of sequencing DNA.
ddNTPs each labeled with different fluorescent dye. Missing the 3′ OH. Sequence terminates when colored label is incorporated. Polymerase products are separated by electrophoresis, labeled ends are detected and sequenced.

Explain the purposes of gene expression studies. Describe the use of DNA microarray assays and explain how they facilitate such studies.
Purpose is to see what genes are active at different stages of development, different tissues, states of health.
Microarrays: Glass slides with 1000s of single strand DNAs. Hybridized with fluorescent cDNA. Determines patterns of expression rapidly.

Explain how in vitro mutagenesis and RNA interference help to discover the functions
of some genes.
To determine functions of unidentifiable genes.
IVM: Disable cloned gene, put it back in organism, observe phenotype.
RNAi: Double-stranded RNA homologous to unknown gene is introduced. Results in breakdown of mRNAs from the gene.

Explain the significance of single nucleotide polymorphisms in the study of the human
Most human variation is due to single base pair differences every 1000 bp. Allows reconstruction of human history, and may determine health, susceptibility, or treatment options.

Describe how genomic equivalence was determined for plants and animals.
Adult cells have same genome as zygote.
In plants, each cell is totipotent.

Describe the general process by which the ewe Dolly and the first mice were cloned.
Egg cells have nuclei removed, Mammary donor cell is arrested in G0, starved causing dedifferentiation. Cells are fused, grown in vitro to early embryo, implanted into surrogate mother.

What problems are associated with animal cloning?
Obesity, pneumonia, liver failure, premature death.
Dedifferentiation doesn’t remove all chromatin modifications.

Describe the two important properties of stem cells. Explain their significance to medicine.
Continually divide, ES stem cells are immortal like cancer cells, differentiate under appropriate conditions, pluripotent but not totipotent.
Significance: Hemocytoblasts in bone marrow give all blood cells. Study differentiation. May be implanted to repair body parts.

Describe how DNA technology can have medical applications in such areas as the diagnosis of disease.
PCR can find the DNA of pathogens in very small amounts, diagnosis hundreds of genetic diseases.
Problem: Some current RFLPs are close to, but not at the abnormal gene.

How is gene therapy accomplished?
Insert RNA for normal gene into retrovirus, allow to infect isolated bone marrow cells. Insert bone marrow cells back into patient.

How are cell cultures used to produce commercial quantities of proteins like insulin?
Make recombinant DNA with desired gene. Put cloned gene into bacteria. Grow bacteria in large quantities.

How are farm animals used to produce commercial quantities of proteins?
Transgenic goats with genes expressed in milk glands produce milk with desired protein.

Explain how DNA technology is used in the forensic sciences.
RFLP can give a probably ID from 1000 cells.
PCR of single tandem repeats is more powerful; more variable and more certain ID, from only 20 cells.

Describe how gene manipulation has practical applications for environmental and agricultural work.
Transgenic organisms used in bioremediation.
Convert of metals to recoverable forms.
Break down harmful organic pollutant.

How are transgenic organisms used in agriculture?
Corn and soybeans with bacterial insecticide genes.
Enviropigs have low P feces.
Golden rice makes Vitamin A.

Describe the safety and ethical questions related to recombinant DNA studies and the biotechnology industry.
New genes in new combinations pollute the gene pool.
Developing technology to treat genetic disease may allow other manipulations