Micro Test #3 (Gene Transfer, Recombinant DNA, & Genetic Engineering)

Give specific (4) examples of genetically engineered crops.

1. Bt as an insecticide: Crops can also be genetically modified to produce this Bt toxin; insects die when they eat the plant. 2. Crop resistance to Roundup (herbicide) 3. Golden rice - genetically engineered to produce vitamin A; 4. Rye has been genetically engineered to resist infection with the fungus Claviceps purpurea, which produces ergot toxin.

What are 2 ways scientists obtain genes they are interested in?

1. Genomic libraries - genome is treated with restriction endonuclease, then RFLP's are inserted into "books" (strains of bacteria, yeast, and bacteriophages) 2. Synthetic DNA - DNA synthesis machines can sequence short strands of DNA (<120 nucleotides long). the sequence must be known before

What are 2 commonly used marker genes?

1. ampicillin resistant gene (beta lactamase gene) - makes bacteria resistant to beta-lactam antibiotics like penicillin. so only bacteria that have the plasmid with this gene will be able to grow on a media that contains amipicillin 2. Beta-Galactosidase (lacZ) gene in the lac operon - the gene of interest is inserted into the lac operon, interrupting the structural gene that code for beta-Galactosidase

What are 2 major goals of genetic engineering using recombinant DNA technology?

1. amplification ( using bacteria to copy a gene because bacteria will copy a gene right along with it) 2. protein synthesis - (make protein coded for on the gene - gene product)

Explain 2 chemical methods of artificial transformation

1. calcium chloride - bathed in calcium chloride the Ca+2 neutralized negative charge in cell envelope so negative charged DNA can come across, then cold shocked then heat shocked. This created a temperature gradient so the outside moves interior and creates fluidity of the plasma membrane 2. Liposome-mediated Transformation - DNA is coated with lipid then fusion of this lipid results in transformation of DNA

Briefly explain the 3 steps of the PCR method.

1. denaturation: heat DNA to near boiling so it denatures 2. Annealing: cool down and add "forward" and "backward" primers (short strands of DNA), primers complimentary based pairs to SS DNA 3. Extension: Warm up add nucleotides and add DNA polymerase III to complete replication

Explain 4 physical methods of artificial transformation.

1. electroporation - briefly shocks cells which form pores in plasma membrane. plasmid DNA can enter through these pores. Natural membrane-repair mechanisms will quickly repair these holes. 2. Microinjection - into animal cells using glass pipette 3. biolistic particle delivery (gene gun) - particles of a heavy metal (ex. gold) are coated with DNA then shot into cell with a burst of helium 4. temperature shock - first cells are incubated on ice then warm bath heat creates temperature gradient and DNA is pulled into the cell.

Why is Bacillus subtilis often preferred over E. coli for making gene product? (2 reasons)

1. its G(+) so no outer membrane and no LPS to act as endotoxin (gene product or protein can be contaminated by endotoxins) 2. its more likely to release its gene product (protein), E. Coli must be lysed to isolate gene product(protein) and LPS from outer membrane might contaminate the gene product(protein)

Identify 2 general methods for identifying transformed bacteria.

1. marker genes - include in plasmid 2. Use radioactive or fluorescent probes that will compliment base pair with the gene of interest.

what is the Southern Blot Method

1. restriction endonuclease is used to cut DNA into smaller fragments call RFLP's 2. RFLP's are eletrophoresed on an agarose to then separate them by size 3. treat agarose gel w/ NaOH to denature DS DNA 4. a sheet of nitrocellulose is places on top of the agarose to transfer DNA to the membrane (DNA is negatively charged, membrane is positively charged) 5. membrane is exposed to hybridization probe - its a sequence of complimentary SS DNA that targets to gene of interest. Its labeled with a chromogenic, fluorescent, radioactive tag. 6. excess probe is washed from membrane and the pattern of hybridization is visualized on x-ray film by audioradiography: radioactive probe UV light: fluorescent probe development of color: chromogenic probe

What are 3 properties of a good plasmid vector?

1. small size (for easier manipulation in lab) 2. circular shape (protects against restriction endonucleases) 3. self replicating (has an origin or replication)

Explain the process of insulin production using recombinant DNA technology.

1. synthetic chains are first constructed to make up two short polypeptide chains that make up the protein 2. each gene is inserted into plasmid vector and linked to the end of the marker gene coded for the bacterial enzyme beta-galactosidase (hormone is co produced from enzyme) 3. polypeptides are recovered, separated from the enzyme, and chemically joined to make insulin

20 cycles of PCR result in how many DNA molecules? (assume you start with 1 molecule)

2^20 = 1,048,576

what is a Bacteriophage

A virus that infects a bacterium

What does Bt stand for?

Bacillus thuringiensis (species of bacteria)

Why can't eukaryotic DNA be put directly into prokaryotic chromosomes?

Because Eukaryotic contains exons and introns. Bacteria only have exons so they don't have the ribozyme enzyme to deal with taking out the introns and splicing the exons together. they don't know what to do with Eukaryotic DNA and no protein synthesis will occur

_________ is the field of DNA sequencing using databases called ___________

Bioinformatics; GenBank.

cDNA & Reverse Transcription (sequence of events)

DNA > pre-mRNA > mRNA > mRNA/cDNA hybrid > denature to get: mRNA and SS cDNA > DS cDNA

What is recombinant DNA?

DNA that contains genes from two different species or organisms

F(+) cells (DO or DO NOT) have pili

DO

What is this RNA's role in gene silencing and RNA interference? (Explain how it works against an RNA virus).

DS siRNA is denatured into two SS siRNA and one is degraded but the one that is complimentary to the viral RNA is kept. When mixed with viral RNA, the SS siRNA binds to the viral and halting it from replication

what does the "F" in F Plasmid stand for?

Fertility

What is a common method of DNA amplification?

PCR (Polymerase chain reaction)

For DNA fingerprinting, what usually must be done to the DNA before using the Southern Blot method?

PCR to amplify the DNA

What does RNAi stand for?

RNA interference

What does RT-PCR stand for? When is RT-PCR used? Explain the process of RT-PCR

Reverse transcription polymerase chain reaction; This is used to amplify RNA sample (like RNA from virus); the process is RNA is converted to cDNA using reverse transcription and reverse transcriptase, since its easier to replicate DNA than RNA. once cDNA the usual process goes as planned.

A common technique for detecting a gene of interest is the

Southern Blot Method

What is the purpose of treating RFLP's with NaOH in the Southern Blot procedure?

This is to denature the DS DNA to make SS DNA so the probe can bind

what is the lysogenic cycle (and 4 examples)

This is when the cycle of the virus becomes latent by becoming integrated into the host chromosomes EX: 1. Chicken pox 2. Human papilloma virus 3. HIV 4. Herpes

what is Transformation

When DNA leaves one bacteria cell and exists in the extra cellular matrix then gets taken up by another bacteria cell and gets put into a chromosomes

Which of the following would represent the ampicillin resistant cells? The ampicillin sensitive cells? (Use an R & an S) a) The cells that didn't grow b) The cells that grew and are blue c) The cells that grew and are white

a) The cells that didn't grow S b) The cells that grew and are blue R c) The cells that grew and are white R

Why isn't bacterial DNA harmed by restriction enzymes?

adenosine or cytosine methylation is part of the restriction modification system of many bacteria. Bacterial DNAs are methylated periodically throughout the genome. A methylase enzyme recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNA's that are introduced into the cell (which are not methylated in this manner) are degraded by the restriction enzyme

Why is it possible to use synthetic genes in the production of insulin?

because the 2 polypeptides chains are only 21 and 30 amino acids long, that means they can be synthesized in the lab.

What does heat do to a double stranded nucleic acid molecule like DNA? what do you call this process?

breaks double bonds between pair nitrogenous bases, separating the two DNA strands; denature

Which of the following would represent the cells that have been transformed by the recombinant plasmid (the desired effect)? a) The cells that didn't grow b) The cells that grew and are blue c) The cells that grew and are white

c) The cells that grew and are white

What does "c" stand for in cDNA? Why is it called that?

complimentary; cDNA is complimentary to mRNA (mRNA acts as template and complimentary base pairing occurs to "build" the cDNA strand)

What was accomplished in the Human Genome Project?

entire human genome was sequenced

Coding sequences of DNA are called

exons

What will be accomplished by the Human Proteome Project?

identifying proteins encoded in genes

Noncoding sequences, once called junk DNA,

includes regulatory sequences, codes for RNA (tRNA & rRNA), telomeres, introns, etc.

what is the lytic cycle

infection of a cell by a virus that leads to replication of a virus and lysis of the host cell. This only occurs if virus is naked if enveloped then virus leaves by budding

Name 2 hormones produced by recombinant DNA techniques

insulin and human growth hormone

DS cDNA can be inserted into bacterial chromosomes because it lacks (introns or exons)

introns

Noncoding sequences of DNA are called

introns

What is the advantage of using a fungus over a prokaryotic organism?

its Eukaryotic so it doesn't have problems with exons and introns its more likely to secrete gene product (protein)

what is a prophage

its is a viral nucleic acid that gets integrated into the bacterium chromosomes. If integrated into a Eukaryotic cell then its called a (Provirus)

what are ribozymes

make mRNA from pre-mRNA by excising introns and splicing together exons

What does DNA amplification refer to?

making multiple copies of DNA

What is the percentage of bacteria that are capable of natural transformation (in the lab)?

only about 1% are naturally "competent"

what is transduction

piece of DNA from one bacteria > into virus capsid > virus infects another bacteria > integrated into bacterium chromosomes. Virus served as vector

what does the "R" is R plasmid stand for?

resistance (antibiotic)

What does RFLP stand for? what enzymes are used to produce it?

restriction fragment length polymorphism; restriction endonuclease.

cDNA is made in a process called _______________, in which DNA is produced from mRNA. What is the name of the enzyme that catalyzes this reaction? What viral family gave us the idea for using this enzyme?

reverse transcription; reverse transcriptase; retroviridae

What is golden rice?

rice that has been genetically engineered to produce vitamin A

Gel electrophoresis is used to separate RFLP's by _________ . The longer the fragment, the _______ the distance it travels in the gel.

size (molecular weight); shorter

What does siRNA stand for?

small interfering RNA

Restriction endonuclease produce staggered cuts, also called

sticky ends

A _________ vaccine is one that consists only of the antigenic protein portion of a pathogen. What is an advantage of the using this kind of vaccine? Give 3 examples of this kind of vaccine.

subunit; no risk of acquiring the pathogen/disease from vaccine; EX. 1. Hepatitis B, 2. Gardasil (against HPV), 3. acellular Pertussis

what is conjugation

the transfer of DNA between bacterial cells using pilli

Explain Blue-White Screening

there are 2 markers included in the plasmid: one for antibiotic resistance and the other for beta-galactosidase which helps hydrolysis of galacotosides. Gene of interest is placed into lacZ so hydrolysis can not be performed. So when on media that contains ampicillin and galactosides. if it doesn't have plasmid at all then it will not be able to grow on media at all. If it does have plasmid, but doesn't have gene of interest that means lacZ will be intact and can hydrolysis galacotosides results in blue colonies color. If it does have the plasmid and gene of interest that means the lacZ is not intacted and cant hydrolysis the galactosides and the colonies are white.

what is Restriction endonuclease and what is it used for?

these are enzymes that are synthesized by bacterial cells that are used to cut and destroy viral DNA. scientist figured out how to use these enzymes for recombining genes.

A ________ is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.

vector

What are 2 common vectors?

viruses (bacteriophages) and plasmids