AP Biology Ch.20 Notes

Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome.
Long DNAs with many genes
Genes make up a small fraction of the genome
Genes are nearly identical to the noncoding sequences
Solutions from DNA technology
Work with small pieces
Many tools for dissecting and analyzing DNA
Can make many copies and express them

How are plasmids used to copy desired DNAs?
Plasmids are naturally occurring small circles of DNA
Can be specifically cut, rearranged and sealed with enzymes
After transformation the bacteria copy the plasmid = gene cloning

Describe the natural function of restriction enzymes.
Made by bacteria to cut up invading DNA
Host DNA protected by methylation at recognition sites

Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule.
Hundreds of restriction enzymes known
Cut at specific sites leaving the same complementary ends
Mixing DNAs cut with the same enzyme allows matching ends to bind
DNA ligase heals backbones to make recombinant DNA

Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid.
Cut plasmid and eukaryotic DNA with the same enzyme
Mix fragments and allow them to match
Transform into bacteria
Identify desired clone
Allow only plasmid-carrying bacteria to grow (AMP)
Add fragments to disrupt an easily screenable phenotype (GLO)

Define and distinguish between genomic libraries using plasmids, phages, and cDNA.
Plasmid library
Thousands of randomly cut pieces inserted into plasmids
Referred to as “bacterial artificial chromosomes”
Phage: thousands of randomly cut pieces inserted into phages
Only contains cloned copies of mRNAs
Isolated mRNA “transcribed” with reverse transcriptase
Then inserted in plasmids or phages

Describe the use of a nucleic acid probe to identify a desired clone.
Probe = short radioactive DNA from the gene of interest
Blot plates holding library to pick up DNA
Put blot in ziplock
Heat to separate DNA strands
Add probe, cool to allow hybridization
Overlay with X-ray film
Develop film, ID plate location of complementary clone

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

Explain how eukaryotic genes are cloned to avoid the problems associated with introns.
mRNAs lack introns
mRNA → cDNA using reverse transcriptase
cDNA inserted into vector

Describe two 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 can be inserted in artificial chromosomes
Can be engineered to perform post-translational modifications

Describe three techniques to aggressively introduce recombinant DNA into eukaryotic cells.
Electroporation opens holes in the membrane with electricity
Micromanipulation with 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.
Quick and more specific and very sensitive
Amplifies only the sequence of interest
Starting mix: Taq DNA polymerase, NTPs, single-stranded DNA primer
Heat to separate target DNA strands
Cool to allow primers to bind to target DNA
Heat-resistant polymerase adds to primer
Heat again, separates new strands
Cool again, polymerase copies again
Result: primer-specified copies double with each cycle
Doesn’t make large quantities of DNA
Errors in the chain reaction are amplified

Explain how gel electrophoresis is used to analyze nucleic acids and proteins and to distinguish between two alleles of a gene.
Electrophoresis separates molecules by size, charge, shape
Nucleic acid analysis
Cut with a specific restriction enzyme → consistent set of pieces
Separate by electrophoresis → consistent pattern of bands
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).
Individuals differ in base sequence
If one DNA differs in a restriction site sequence it will not be cut
A different pattern of bands will result
Process of Southern blotting
Cut with restriction enzymes, separate pieces in a gel
Blot, etc. as described above

Describe the dideoxy chain termination method of sequencing DNA.
Starting mix: DNA, DNA polymerase, dNTPs, single-stranded DNA primer
Crucial ingredient
ddNTPs each labeled with a different fluorescent dye
ddNTPs are missing the 3′-OH
Chain terminates when the colored label is incorporated
DNA polymerase products are separated by electrophoresis
Labeled ends are detected

Explain the purposes of gene expression studies. Describe the use of DNA microarray assays and explain how they facilitate such studies.
See what genes are active at different stages of development
… in different tissues
…in different states of health
= glass slides printed with dots of 1000s of single-strands DNAs
Hybridized with fluorescently labeled cDNA
Rapidly determines patterns of expression

Explain how in vitro mutagenesis and RNA interference help to discover the functions of some genes.
Especially useful for determining functions of unidentifiable genes in vitro mutagenesis
Disable the cloned gene
Put it back in the organism, observe phenotype
RNA interference
Double-stranded RNA homologous to the unknown gene is introduced
Results in the breakdown of the mRNAs from the gene

Explain the significance of single nucleotide polymorphisms in the study of the human genome.
Most human variation is due to single base differences
= 1 in 1000 bp = 3 million differences
Allows reconstruction of human history
May determine health, susceptibility, treatment options

Describe how genomic equivalence was determined for plants and animals.
Genomic equivalence = adult cells have the same genome as the zygote
Cloning Plants: Single-Cell Cultures
Plant tissue culture
Single plant cell can grow into an adult
Each cell is totipotent, can form all adult cell types
Cloning Animals: Nuclear Transplantation
Frog cloning
Kill the egg nucleus with UV
Replace it with another nucleus
Early embryonic nuclei give the best results
Tadpole intestinal cell nuclei work 2% of the time

Describe the general process by which the ewe Dolly and the first mice were cloned.
Mammary donor cells grown in vitro were arrested in G0
Egg cells had their nuclei removed
Electric current was used to fuse the two cells
Grown in vitro to early embryo stage
Implanted into a surrogate mother

What problems are associated with animal cloning?
Often have 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
Embryonic stem cells are immortal, like cancer cells
Under appropriate conditions will differentiate
Pluripotent but not totipotent
Significance to medicine
For transplantation, hemocytoblasts in bone marrow give all blood cells
Gives a way to 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
Diagnose hundreds of genetic diseases
ID genetic diseases in embryos produced in vitro
Problem: some current RFLP markers are close to, but not at the abnormal gene

How is gene therapy accomplished?
Insert RNA for the normal gene into retrovirus
Infect isolated bone marrow cells
Allow retrovirus to insert the normal gene into chromosome
Inject bone marrow cells back into patient

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

How are farm animals used to produce commercial quantities of proteins?
Transgenic goats with genes expressed in milk glands
Desired protein is purified from milk

Explain how DNA technology is used in the forensic sciences.
RFLP analysis can give a probable ID from 1000 cells = DNA fingerprint
PCR of single tandem repeats is more powerful
More variable → more certain ID
Requires only 20 cell sample
Both require accompanying statistical arguments

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 pollutants

Describe how plant genes can be manipulated using the Ti plasmid carried by Agrobacterium as a vector.
a. A segment of this plasmid is inserted into the host chromosome
b. Desired new DNA is added to the Ti plasmid at that point
c. Put back into Agrobacterium for infection or inserted directly
d. Only works in dicots

How are transgenic organisms used in agriculture?
Corn and soybeans with bacterial insecticide genes
Enviropigs: have an added enzyme that gives them 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 can pollute the gene pool
Developing the technology to treat genetic disease may allow other manipulations

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