SA4225-Small Animal Research Techniques (SART) Exam 2 Study Guide Spring 2025

These flashcards cover key concepts and questions from the SA4225-Small Animal Research Techniques (SART) course to aid in exam preparation.

What is the purpose of DNA extraction?

To isolate DNA from cells for analysis.

What are the four steps common to all types of DNA extraction methods?

Cell lysis, precipitation, washing, and rehydration.

Describe the basic technique behind the preparation of crude lysates in DNA extraction.

Cells are lysed to release DNA without extensive purification.

What are the disadvantages of organic extraction methods in DNA extraction?

They can be toxic, labor-intensive, and may co-extract impurities.

What are the advantages of silica-based methods of DNA extraction?

They are quick, efficient, and can yield high-quality DNA.

What is the purpose of RNA isolation?

To extract RNA for analysis, particularly mRNA.

What is the main concern when isolating RNA molecules compared to DNA molecules?

RNA is more prone to degradation by RNases.

Which type of RNA represents gene expression?

Messenger RNA (mRNA).

What are Rnases?

Enzymes that degrade RNA.

What are the basic steps in RNA isolation methods?

Lysis, phenol-chloroform extraction, precipitation, and resuspension.

What is gel electrophoresis?

A laboratory technique used to separate DNA, RNA, or proteins based on size.

What is the purpose of gel electrophoresis?

To visualize and analyze nucleic acids or proteins.

What kind of gel is used in DNA electrophoresis?

Agarose gel.

What is blue tracking dye used for in gel electrophoresis? Does it bind DNA?

It is used to monitor the progress of the sample during electrophoresis, and it does not bind DNA.

Explain how DNA moves through the gel during electrophoresis.

DNA is negatively charged and moves towards the positive electrode.

What is ethidium bromide?

A DNA intercalating agent used to visualize DNA in gels.

What is the purpose of the southern blot?

To detect specific DNA sequences in a sample.

Name and describe the 5 steps of the southern blot technique.

1. Digestion of DNA, 2. Gel electrophoresis, 3. Transfer to membrane, 4. Hybridization with probe, 5. Detection.

What information does a southern blot provide?

It provides information on the presence and size of specific DNA sequences.

How is the probe for southern blot designed?

It must be complementary to the target DNA sequence.

What is HRP in the context of southern blotting? How does it provide a detection signal?

Horseradish peroxidase; it catalyzes a reaction that produces a detectable signal.

What is the purpose of Northern blotting?

To detect and analyze specific RNA sequences.

What are the basic steps of the Northern blot?

1. RNA extraction, 2. Gel electrophoresis, 3. Transfer to membrane, 4. Hybridization with probe, 5. Detection.

What information does Northern blotting provide?

It provides information on the size and expression levels of specific RNA.

What is the purpose/theory behind PCR?

To amplify specific DNA sequences.

Who invented PCR?

Kary Mullis.

What kind of information does PCR provide?

It provides quantities of specific DNA sequences.

What are primers and why are they necessary in PCR?

Short sequences of nucleotides that initiate DNA synthesis.

What are the necessary ingredients of a PCR reaction?

DNA template, primers, nucleotides, and DNA polymerase.

Name and describe the three main steps of PCR.

1. Denaturation, 2. Annealing, 3. Extension.

What is a thermocycler?

A machine that automates the temperature changes required for PCR.

What is Taq polymerase and what is the significance of this discovery?

A heat-stable DNA polymerase essential for PCR.

What is the purpose/theory behind Real-time PCR?

To quantify DNA in real time during amplification.

What is reverse transcriptase?

An enzyme that converts RNA into complementary DNA (cDNA).

What is cDNA?

Complementary DNA synthesized from an RNA template.

Describe the steps to make cDNA.

1. Isolate RNA, 2. Add reverse transcriptase and primers, 3. Synthesize cDNA.

What are the differences between RT-PCR, PCR, and qPCR?

RT-PCR involves RNA to cDNA conversion, PCR amplifies DNA, and qPCR quantifies DNA in real time.

What are antibodies?

Proteins produced by the immune system to identify and neutralize foreign objects.

How are antibodies made?

By B cells in response to an antigen.

What is an epitope?

The specific part of an antigen that an antibody binds to.

What is the difference between monoclonal and polyclonal antibodies?

Monoclonal antibodies are identical and target a single epitope; polyclonal antibodies are a mixture from multiple B cells.

How are monoclonal and polyclonal antibodies made?

Monoclonal antibodies are produced from a single B cell clone; polyclonal antibodies are produced from different B cell clones.

What are the advantages and disadvantages of monoclonal antibodies?

Advantages: specificity; Disadvantages: cost and complexity.

What is the purpose of immunoprecipitation?

To isolate and concentrate specific proteins from a mixture.

What are the basic steps of immunoprecipitation?

1. Cell lysis, 2. Incubation with antibody, 3. Capture beads, 4. Washing, 5. Elution.

What is the purpose of immunohistochemistry?

To visualize specific proteins in tissue sections.

What are the differences between direct and indirect immunofluorescence methods?

Direct uses a labeled primary antibody; indirect uses a labeled secondary antibody.

What is the purpose behind Western blotting?

To detect specific proteins in a sample.

What are the steps involved in Western blotting?

1. Gel electrophoresis, 2. Transfer to membrane, 3. Blocking, 4. Incubation with antibodies, 5. Detection.

What kind of information does Western blotting provide?

It provides information on protein size, abundance, and post-translational modifications.

Describe the importance of the stacking gel in Western blotting.

It concentrates and positions proteins in a tight band before separation.

How is the secondary antibody detected in Western blotting?

Through enzyme-linked or fluorescent labeling.

What are the basics of ELISAs?

Enzyme-linked immunosorbent assays for detecting antigens or antibodies.

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay.

What are the basic steps of an ELISA?

1. Coating with antigen, 2. Blocking, 3. Adding sample, 4. Detection with antibodies, 5. Substrate addition.

What are the differences between direct, indirect, and competitive ELISAs?

Direct uses a single antibody; indirect uses a secondary, and competitive measures bound vs unbound.

What is a standard curve in ELISA?

A graph showing the relationship between known concentrations and signal.

How do you make a standard curve and what information does it provide?

Through serial dilutions; it quantifies unknown samples based on absorbance.

What are transgenic mice used for?

To study gene function and model human diseases.

What is superovulation? How is it achieved?

Induction of multiple ovulations using hormonal treatments.

What is a stud male and how are they managed?

A male used for breeding, managed for health and fertility.

What is the GOI?

Gene of Interest.

What is a promoter?

A DNA sequence that initiates transcription of a gene.

What is a reporter gene and how is it used?

A gene that encodes for an easily measurable product used to indicate activity of another gene.

What are the important stages of embryo development?

Zygote, morula, blastocyst.

What does pluripotent mean?

The ability of a cell to develop into multiple cell types.

What is the Whitten effect?

Synchronization of estrous cycles in female mice resulting from male presence.

What are embryo recipients?

Surrogate mothers that carry implanted embryos.

What are the steps of designing and generating a transgene?

1. Identifying GOI, 2. Cloning, 3. Insertion into a vector, 4. Transfection into host cells.

What are plasmids and how are they used?

Circular DNA molecules used as vectors to carry genetic material.

What are the steps for gene insertion using pronuclear injection?

1. Micromanipulation of zygotes, 2. Injection of DNA, 3. Implantation into surrogate.

What are founders in transgenic research?

The first generation of transgenic animals used for breeding.

What are the strategies used when mating chimeric mice?

Using males with excellent breeding potential to produce transgenic offspring.

What are conditional knockouts?

Knockouts that can be activated or deactivated at certain ages or conditions.

What is gene editing? How does it differ from gene targeting?

Gene editing involves directly altering nucleotide sequences; gene targeting involves replacing or removing them.

What are the advantages of gene editing?

Precision, efficiency, and versatility in genetic modifications.

What is CRISPR/Cas9?

A revolutionary gene editing technology using a specific RNA guide.

What is the COIN system?

A system used for efficient gene integration in mouse models.

What are Floxed Stop Cassettes?

Genetic elements used for conditional gene expression.