Recombinant DNA Technology - Microbiology - Chapter 8 Bauman

What provides the energy for DNA polymerization in a PCR reaction?

Deoxyribonucleoside triphosphates

What is the most convenient vector for creating recombinant DNA?

Viruses

When are blunt ends used?

When 2 organisms do *not* conveniently have the same restriction sites.

What are DNA molecule immobilized on in Southern blotting?

nitrocellulose membranes

The natural role of restriction enzymes in bacteria is to...?

protect the cell from invading phages.

Vector

"a carrier for the gene that researchers want to introduce into an organism." -which are nucleic acid molecules, such as viral genomes, transposons, and plasmids. -Potential Application: Altering the genome of a cell.

Polymerase Chain Reaction

*PCR* -allows researchers to replicate a large number of identical molecules of DNA rapidly, in vitro. -Potential Application: multiplying DNA for various applications

Biotechnology

*The use of organisms to make products.* Utilizes many different techniques that allow biologists to manipulate the genomes of cells. Ex: bread, wine, beer, cheese, etc.

What is Agarose? How does it work?

-A purified sugar component of agar. -Acts as a molecular sieve that retards the movement of DNA fragments by size.

Gel Electrophoresis - Steps

-Agarose gel slab loaded w/ DNA w/ micropipette -Gel put into a electrophoresis chamber that contains a liquid salt buffer -Electricity turned on

Gel Electrophoresis

-Technique of recombinant DNA technology. -Separates DNA molecules by size, charge, or shape. -Composed of agarose. -Potential Application: Separating DNA fragments by size *aka Genetic or DNA Fingerprinting*

Recombinant DNA Technology

-Type of biotechnology in which scientists *change the genotypes and phenotypes of organisms.* -Not a single procedure/technique but rather a collection of tools and techniques scientists use to manipulate the genomes or organisms.

Sticky Ends

-Type of restriction enzyme -Makes staggered cuts of the 2 strands of DNA, producing fragments that terminate in sticky ends. -Bind only to complementary, sticky-ended fragments produced by the same restriction enzyme.

Blunt Ends

-Type of restriction enzyme -When both strands of DNA are cut at the same point. -More difficult to make -Advantage: Non-specific and can be combined easily

In which direction does DNA polymerase synthesize the new DNA strand?

5' to 3'

cDNA

"complementary DNA" DNA synthesized from an mRNA template using reverse transcriptase.

Reverse Transciptase

-Complex enzyme -Allows retroviruses to make dsDNA from RNA templates -Used in recombinant DNA technology to make cDNA.

Gene Therapy

-Cures various diseases by replacing defective genes with normal genes. -The use of recombinant DNA technology to insert a missing gene or repair a defective gene in human cells.

Southern Blot

-Technique of recombinant DNA technology. -A procedure that transfers DNA from an agarose gel to a nitrocellulose membrane, which are more durable. -DNA on membrane can be incubated with a single-stranded DNA "probe" to look for the presence of a specific gene sequence.

PCR - Involves reagents/tools

-Thermocycler -Target DNA -Taq DNA Polymerase -Nucleotides -Primers

Put the following events in the construction of a recombinant DNA molecule in the correct order: a. ligation of desired gene to plasmid DNA b. introduction of the plasmid into bacteria c. restriction enzymes cut gene of interest and plasmid DNA d. growth of cells on an antibiotic-containing medium

C, A, B, D

What attaches the target gene to a desired location?

DNA Ligase

What if a researcher needs to determine the info for thousands of genes, what method would they use?

DNA Microarray

What if the researcher just wants to know if the gene is being expressed (transcribed or translated)?

DNA Microarray or FISH - Fluorescent in-situ Hybridization if finding the exact location of the gene is unnecessary

How does gel electrophoresis work with DNA?

DNA has an overall negative charge and moves toward the positively charged end of the electrophoresis box. Separated DNA fragments don't have to remain in the gel. There are always ways of removing tem for further study.

What if a bacterium can't be made competent, or the multicellular organism has an immune system? Name some methods we could use around this.

Electroporation Protoplast Fusion Gene Gun Microinjection Use viruses to deliver DNA DNA Sequencing

Name some artificial methods to introduce DNA into cells.

Electroporation Protoplast Fusion Injection

Restriction Enzymes

Enzymes that cut DNA at specific nucleotide sequences -Used to produce recombinant DNA molecules. -Cut DNA only at restriction sites

Give some examples of Transgenic Organisms

Herbicide resistance Tolerance to salty soils Resistance to freezing and pests Improvements in nutritional value and yield

How are restriction enzymes categorized?

How they cut DNA strands 1. Sticky Ends 2. Blunt Ends

Why would a recombinant DNA molecule be inserted into a host cell?

It can be copied, transcribed, and translated into a desired protein.

Why is DNA polymerase from Thermus aquaticus ideal for PCR?

It can withstand the high temperatures associated with PCR.

What are some Biotechnology uses?

Medicine Finding new microbes Agriculture

What tools of recombinant DNA technology does it include?

Mutagens Reverse Transciptase Synthetic Nucleic Avids Restriction Enzymes Vectors

Give an example of restriction enzymes in nature.

Provide bacteria w/ some protection against bacteriophage infection (by cutting up phage DNA once it is injected into a bacterium)

Transgenic Organisms

Recombinant plants and animals that have been altered for specific purposes by the addition of genes from other organisms. -aka GMO (Genetically Modified Organisms)

Which of the following enzymes can make specific cuts in DNA molecules?

Restriction Enzymes

Synthesis of cDNA requires the use of what?

Reverse Transcriptase

Gel electrophoresis will separate DNA fragments, but what if a researcher want to locate a particular gene, what method do they use?

Southern Blot

A researcher mistakenly uses a heat-sensitive version of DNA polymerase in a PCR. What would occur?

The PCR will stop after one cycle.

How do restriction enzymes cut DNA sequences?

They cut DNA at sites, called recognition sites, that have specific nucleotide sequences.

What is the function of the primers in PCR?

They provide a 3' end for the DNA polymerase.

What does recombinant DNA technology require?

a VECTOR.

Genetic Fingerprinting involves what?

aka DNA Fingerprinting 1. Procure a sample of DNA. 2. Making multiple copies of it via PCR. 3. Cutting the copies w/ restriction enzymes. 4. Separating the fragments by gel electrophoresis to produce a unique pattern.

Which of the following would be a useful phenotypic marker on a vector?

antibiotic resistance gene

Xenotransplants

are animal cells, tissues, or organs introduced into the human body.

DNA Microarray

-tool of biotechnology *-Reveals presence of specific DNA or RNA molecules in a sample.* -Numerous distinct ssDNA molecules bound to a substrate and used to probe for complementary sequences. -Uses probe-labeled DNA stuck to special glass slides -When a researcher needs to probe for tens of thousands of genes!

PCR - Steps

1. Denaturation - Separates 2 strands of the target DNA 2. Priming - bind to complementary sites on the target DNA providing Taq DNA Polymerase w/ a starting point 3. Extension - Taq DNA Polymerase binds to the target DNA next to the primers and synthesizes new DNA strands.

When would you use DNA Microarrays?

1. Monitoring gene expression 2. Diagnosing infection 3. Identifying organisms in an environmental sample

Describe how a plasmid works as a vector.

1. Plasmid is isolated. 2. DNA is cleaved by an enzyme into fragments (DNA containing gene of interest). 3. Gene is inserted into plasmid. 4. Plasmid is taken up by a cell such as a bacterium. 5. Cells with gene of interest are cloned.

Useful properties of Vectors.

1. Small-->to allow manipulation in a lab 2. Survive inside cells 3. Need a recognizable genetic marker 4. Ensure genetic expression (by providing all necessary info for the marker gene)

Synthetic Nucleic Acid

DNA molecule prepared in vitro Potential Application: Creating DNA probes to localize genes within a genome

In general, how might recombinant DNA technology be used to prevent a genetic disorder caused by a mutation in a single gene?

To insert a desirable gene, remove an undesirable gene, or replace a defective gene with a functioning gene