Chapter 20: Biotechnology

What two techniques are used in performing a Southern blot?

combines gel electrophoresis and nucleic acid hybridization

What are some drawbacks to PCR?

despite its speed and specificity, occasional errors occur during PCR and it has a limit to how many good copies can be made. Problems arise when large amounts of DNA are desired or when the DNA fragments themselves are very long.

What was the first technique developed to sequence DNA?

dideoxyribonucleotide chain termination method

Define genetic engineering

direct manipulation of genes for practical puropses

Why do clones not always look or behave exactly the same?

environmental influences, methylation patterns, random X chromosome inactivation

What are some problems associated with animal cloning?

epigenetic changes in the chromatin cause proteins that need to be expressed in embryos not to be expressed, explains improper embryo development. DNA in cells from cloned embryos have unusually high amounts of methylated DNA.

What methods to scientists use to determine gene function?

in vitro mutagenesis, RNA interference, SNP study

What is in vitro mutagenesis?

involves mutating a gene to destroy or alter its function and observe the effects it has when returned to the cells.

What is the purpose of a genomic library?

it can be used for research, sequencing, or commercial purposes. "the complete set of plasmid containing cell clones".

What can cDNA not be used for in terms of information storage

It cannot be used to create a complete genomic library because it is derived from mRNA and only contains exons not the complete genome

Why does a dideoxyribonucleotide terminate a growing DNA strand?

It lacks a 3' -OH group which is where the next nucleotide is supposed to attach

Explain the 4 steps of a DNA microarray assay

1. isolate mRNA 2. Make cDNA by reverse transcription using fluorescent tagged nucleotides 3. Apply cDNA mixture to array, cDNA hybridizes with any complimentary DNA on microarray 4/ Rinse off excess cDNA, scan microarray for fluorescence. Yellow spots indicate that the gene is expressed in the tissue sample

How many molecules will be produced by four PCR cycles?

2^n , so 2^4= 16 molecules

Explain the four basic steps of gene clothing

1. Gene is inserted into plasmid 2. Plasmid put into bacterial cell 3. Host cell grown in culture to form a clone of cells containing the cloned gene of interest 4. basic research and various applications.

Explain the six steps in reproductive cloning for mammals

1. cultured mammary cells are semi starved, arresting the cell cycle and causing dedifferentiation. 2. the nucleus of an egg cell is removed 3. the cells are fused together 4. grown in culture 5. implanted in uterus 6. embryonic development and birth to clone

What are two problems with bacterial gene expression systems?

1. gene expression varies between eukaryotes and prokaryotes, need to insert expression vector to serve as a promoter to ensure foreign gene is expressed 2. Eukaryotes have introns and this prevents correct expression in bacteria because they do not have RNA splicing machinery. to solve this, could insert cDNA since it only has exons. Or a better solution is usually to use yeast since they are eukaryotic and produce quickly like bacteria.

What is RT-PCR?

A quicker and more sensitive method for Northern blotting. Reverse transcriptase- Polymerase Chain Reaction is done on mRNA and results in cDNA that is then amplified. the copies are then run on gel electrophoresis and observed

What polymerase is used in PCR?

An unusually heat stable polymerase called taq polymerase. Found in hot spring bacteria if any other polymerase was used for PCR it would be denatured with the DNA in the first step.

Explain how transgenic "pharm" animals might be able to produce human proteins

DNA for a foreign gene is injected into the nucleus of a fertilized egg in vitro. Animal grows and expresses the gene by producing the protein that is coded for

Define recombinant DNA

DNA formed with segments from two different sources

Why is the DNA sample to be separated by gel electrophoresis always loaded at the cathode/negative end of the power source?

DNA has a negative charge and is repelled by the negative cathode and attracted to the positive anode. When placed at the negative side, the sample moves towards the positive end, separating the molecule

What is the major difference between embryonic stem cells (ES) and adult stem cells?

Embryonic stem cells can generate all embryonic cell types, are from blastocyst stage of embryo Adult stem cells come from adult cells such as in one marrow, they can generate a limited number of cell types. these stem cells are not yet fully understood

What does pluripotent mean?

Ex. Embryonic stem cells. they are capable of differentiating into many different cell types but cannot form an entire organism like totipotent.

How does the Ti plasmid make genetic engineering in plants a possibility?

It is from a soil bacterium that integrates a specific segment of its DNA into the host cells DNA. This certain segment can be modified to contain the desired gene.

What are genetically modified organisms, and why are they controversial?

Food that has been modified with foreign genes to have more desirable traits for production. They are controversial for many reasons. They might spread their genetic modifications to their wild relatives causing super weeds, humans may have allergic reactions, getting rid of diversity, have unknown effects on humans, etc.

Explain the idea of gene therapy, and discuss the problems with this technique as demonstrated in the treatment of SCID

Gene therapy is introducing genes into an afflicted individual for therapeutic purposed. Normal allele is inserted into the affected somatic cells. Excellent in theory but has some problems in practice. In the patients being treated with gene therapy for their SCID, 3 developed leukemia. It is possible that this happened because the DNA was injected close to a gene that codes for proliferation of blood cells, it is not always controlled where the new DNA is inserted and this can have disastrous effects on the genome

Explain how microarrays are used in understanding patterns of gene expression in normal and cancerous tissue

Microarray can be performed on normal cells and then compared to a microarray of cancer cells, or even done simultaneously. This allows us to see what proteins cancer cells are expressing that normal cells are not and vice versa. This gives us an idea of what goes wrong in gene expression in cancer cells

Explain nucleic acid hybridization

Nucleic acid hybridization uses a RNA or DNA molecule as "probe". It is complimentary to a known sequence of the gene of interest. It binds to this gene and because the nucleic acid is radioactively labeled, the genes location can be determined

A small sample of dodo bird DNA is found, what technique should be used to increase the amount of the DNA for testing to be done?

Polymerase Chain Reaction (PCR) to amplify the genes

Briefly explain the process of PCR

Polymerase Chain Reaction. 1. Heat briefly to separate strands 2. cool to allow primers to form and hydrogen bonds with ends of target sequences (annealing stage) 3. DNA polymerase adds nucleotides to the 3' end of each primer (extension)

Explain how restriction enzymes work

Restriction enzymes cut very specific sequences on a gene, when it cuts it cuts in a staggered way that creates sticky ends. Then new complimentary DNA cut by the same restriction enzyme can come in and bind, DNA ligase acts a glue to piece the new DNA together.

Why are SNP's useful when observing a gene?

SNPS are typically inherited with certain alleles. When two people share a disease and have the same SNP that unaffected people dont have, it can indicate where to look in the genome to find a mutation in the actual gene/allele.

Explain how single nucleotide polymorphisms (SNPs) and restriction fragment length polymorphisms (RFLPs) were demonstrated in analyzing sickle cell alleles

SNPs cause a restriction enzyme to cut differently since the specific sequence may be repeated more or less between cells. This results in different restriction fragment length polymorphisms. When run on the gel, we see different banding patterns because of these SNPs and consequent RFLPs.

How did Dolly the sheep die and what does this indicate about reproductive cloning?

She died from illnesses commonly associated with older sheep. This indicates that the age of the donor cells affects the age of the new cells. Something in the nucleus is affected after differentiation and during reproductive cloning it can not be completely reprogrammed, causing trouble in cloned organisms.

Describe how short tandem repeats can produce a sensitive genetic profile.

Short tandem repeats are repeats of certain sequences before and after the actual allele coded for in a gene. They are often inherited with the allele. Based on the number of short tandem repeats present in a person, a profile of the entire genome is very unique and no one else will all of the same short tandem repeats as a person except for in the case of identical twins.

Plasmids are important in biotechnology. Give a full and complete definition of a plasmid.

Small circular DNA molecules that replicate separately from the bacterial chromosome. Contain a small number of genes that a bacteria may or may not need for certain environments

How are small molecules used as pharmaceutical drugs?

Small molecules such as imatinib inhibit the overexpression of a specific leukemia causing receptor

A patient who is a carrier for sickle cell anemia would have a gel electrophoresis pattern showing four bands, explain why.

The banding pattern depends on how many times the restriction enzyme can locate the specific sequence that it cuts at. For a normal patient, there are 3 fragments, for a mutant there are 2. There are four for the carrier because it is cut at all the places the mutant AND the normal is cut at . (one allele of each)

Explain why shorter DNA molecules travel farther down the gel than larger molecules

The gel acts as an obstacle course, the agarose fibers impede larger molecules. The smaller molecules can move more quickly through the gel.

How is nuclear transplantation performed in animals?

The nucleus of an unfertilized or fertilized egg is removed and replaced with the nucleus of a differentiated cell

Define gene cloning

The production of multiple copies of a single gene

Why are some colonies white? Why is this important?

These cells have not taken up the recombinant plasmid. This is evident because they do not have the enzyme to break down x-gal and form a blue product

How might induced pluripotent stem cells (iPS) resolve the debate about using stem cells for medical treatments?

They behave similarly to stem cells but are still relatively not understood. Cells are taken and reverted into stem cells instead of being harvested from human embryos.

Explain why a genetically engineered plasmid would be engineered with ampicillin resistance and the lacZ gene?

This provides two sources of proof that the bacteria has in fact taken up the plasmid if the bacteria A: grows on a plate of ampicillin and B: appear blue because of the product of hydrolyzing x-gal with the lacZ gene

What is electroporation?

a method to introducing recombinant DNA into eukaryotic cells. Brief electrical pulse is applied, creating temporary holes in the plasma membrane

In recombinant DNA, the term vector can refer to___

a plasmid used to transfer DNA into a living cell

What is gel electrophoresis?

a technique used to separate nucleic acids or proteins that differ in size or electrical charge

What is a totipotent cell?

any cell with the ability to dedifferentiate and give rise to all the specialized cell types of the organism. Can be an entire organism. these are the types of cells the first 4 days after an egg is fertilized

Explain how a bacterial artificial library (BAC) and cDNA library are formed

large plasmids are trimmed down to contain and store many genes. cDNA libraries are derived from the mRNA, store cDNA which only entails exons. not a complete genome

Expression of a cloned eukaryotic gene in a bacterial cell involves many challenges. The use of mRNA and reverse transcriptase is part of a strategy to solve the problem of ____?

post-transcriptional processing (bateria dont have these capabilities like eukaryotes)

What kind of applications can genetic modifications have?

protein producing animals, environmental cleanup, improve agricultural products

What are stem cells?

relatively unspecialized cells that can reproduce indefinitely and under the right conditions differentiate into specialized cells.

What is Northern blotting?

similar to southern blotting except gel electrophoresis is performed on samples of mRNA

Why are the four nucleotides in DNA each labeled with a different color of fluorescent for DCTM?

so the colors can be used to identify which nucleotide was added and ultimately sequence the entire DNA strand

Describe how a radioactively labeled nucleic acid probe can locate the gene of interest on a multiwell plate

the DNA is denatured in a nylon membrane. The nucleic acid probe binds to the gene of interest creating a double stranded DNA again. Then it is laid under photographic film and the radioactive label turns black. From this the well location of the gene of interest can be determined.

Define biotechnology

the manipulation of organisms or their components to make useful products

What is a cloning vector?

the original plasmid, the DNA molecule that can carry foreign DNA into a host cell and replicate it there

After transformation has occurred, why are some of the colonies blue?

they have the lacZ gene which codes for an enzyme that hydrolyzes x-gal and creates a blue product

What is the purpose of a Southern blot?

to detect and identify only the genes of interest on a gel

Plants are more readily manipulated by genetic engineering than animals because ___?

unlike somatic animal cells, a somatic plant cell can often give rise to a complete plant

What is in situ hybridization?

use of fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism.

Define polymorphisms

variations in DNA sequence among a population