Molecular Bio Exam 2

What do Southern Blots, Northern blots, and Western blots run?

• Southern: Runs DNA gels

• Northern: Runs RNA gels

• Western: Runs protein gels

What is the probe for a western blot called?

The antibody.

What is a probe?

A single-stranded DNA (ssDNA) or RNA (ssRNA) labeled with a tracer (radioactivity, hapten, or fluorophore) that allows tracking of probe hybridization to unknown DNA or RNA. 25-1000 bp length

What is the process of “random primed labeling”? What is the benefit of this technique

1. Denature the DNA template

2. Annealing: hexamers bind to single-stranded DNA

3. Extension: primers are extended with Klenow fragments

4. Denature the strands again and use single strands as probes

Results in uniform labeling of DNA probe; is faster and more specific than nick translation.

What are the two fragments of E.coli DNA polymerase I and their functions?

• Klenow fragment: results from enzymatic cleavage of DNA pol I by protease subtilisin.

  • 5’ to 3’ polymerase activity: synthesizes new DNA strands

  • 3’ to 5’ exonuclease activity: proofreads and corrects mismatched bases.

• Other subunit

  • 5’ to 3’ exonuclease activity: removes RNA primers or damaged DNA strands.

  • Allows for “nick translation”

Explain the 6 steps of the Klenow fill-in method.

• DNA Digestion: Restriction endonucleases cut the dsDNA

• Overhang: This leaves a 5’ single-stranded overhang

• Enzyme addition: Klenow polymerase is added

• Nucleotide incorporation: Labeled and unlabeled dNTPs are incorporated

• Fill-in synthesis: Klenow extends the recessed 3’ end

• Probe labeling: The final fragment carries labels on its 3’ end

What enzyme is responsible for 5’ end labeling and what does it add?

• Enzyme: T4 polynucleotide kinase

• Transfers the y-phosphate from ATP to the 5’ end

What enzyme is reponsible for 3’ end labeling and what does it add?

• Enzyme: Terminal transferase

• Can add several modified dNTPs to the 3’ end.

What enzymes are used for in vitro transcription, and what are the benefits?

Enzymes: Phage SP6, T7, or T3 RNA polymerase

• Results in uniform labeling of RNA probe

What are the types of tracers and how are they detected?

1. Radioactivity

2. Hapten

3. Biotin

4. Fluorophores

Autoradiography, antibodies or streptavadin.

What are the steps of a southern blot?

1. Digest DNA

2. Gel electrophoresis

3. Transfer to nylon membrane

4. Hybridization

5. Exposure

What are the pros and cons of southern blots?

Pros

• Long and complex DNA analysis

• Multiplex capability

Cons

• Time-consuming and Labor-intensive

• High DNA input requirement

What are the steps of northern blotting?

1. RNA separation by gel electrophoresis

2. Blot on nitrocellulose

3. Label with probe

4. Use X-ray film to detect the probe

What are the pros and cons of northern blots?

Pros

• High specificity

• Reusable membranes

Cons

• High risk of RNA degradation

• Low throughput

For IF (immunofluorescence),

1. What does it measure?

2. What are the controls/how does it work?

1. Tells you where a protein is and how much of it is present.

2. Controls include no primary control, using an isotype control, using a positive control, and a negative control.

• Fixation/Permeabilization: Cells are frozen and fixed, then treated with a detergent to poke holes in the membrane so antibodies can enter.

• Primary antibody: An antibody made to bind to the target protein is added.

• Secondary antibody: A second antibody linked to a fluorophore is added that binds to the primary antibody.

• Visualization: The sample is exposed to specific wavelengths of light under a fluorescence microscope, showing where the protein is.

What is the difference between direct IF and indirect IF?

Direct IF skips the secondary antibody, and the fluorophore connects directly to the primary antibody. Indirect IF, which uses a secondary antibody, is more common because it amplifies the signal.

For ELISA (enzyme-linked immunosorbent assay),

1. What does it measure?

2. What are the controls/how does it work?

1. Measures the presence and concentration of specific antigens or antibodies in a liquid sample.

• Immobilization: The antigen or antibody is attached to the surface of a 96-well polystyrene plate.

• Binding: The sample is added to the wells, and if the target molecule is present, it binds to the matching antigen or antibody.

• Detection: An enzyme-linked detection antibody is added to bind the target complex.

• Signal Generation: A chemical substrate is added, and the enzyme converts the substrate into a luminescent product.

• Measurement: A microplate reader measures the signal intensity.

2. Controls include positive controls, negative controls, blank controls, standard controls, and spike controls.

For FISH (fluorescence in situ hybridization),

1. What does it measure?

2. What are the controls/how does it work?

1. Measures the presence, location, number, and arrangement of specific mRNA sequences inside cells.

• Fixation/permeabilization

• Hybridize: The sample and probes are combined, and fluorescent probes bind to the corresponding sequence.

• Wash: Unbound probes are washed away.

• Imaging: The slide is viewed under a fluorescent microscope.

2. Controls include internal control probes, a positive control, and a negative control.

What are the 3 limitations of DNA Polymerase enzymes?

1. Add dNTPs growing molecule

2. Needs a template

3. Can only add to the 3’ end

2 main functions of DNA polymerase III?

1. DNA replication (5’ - 3’ polymerase)

2. DNA repair (3’ - 5’ exonuclease)

3 functions of DNA polymerase I?

1. DNA replication

2. DNA repair

3. Okazaki fragment maturation (5’ - 3’ exonuclease)

What are DNA polymerade II, IV, and V responsible for?

TLS (trans lesion synthesis).

Why is the origin of replication (oriC) in bacterial chromosomes AT rich?

Why is the origin of replication negatively supercoiled?

AT only has 2 hydrogen bonds, so it is easier to open up for replication.

Negative supercoiling is less tightly coiled, so it is easier to open up for replication.

What are the 7 components of a replisome, and what is their function?

1. Primase: adds RNA primers to DNA strands

2. Helices: opens up the two strands of DNA

3. DNA Pol III

4. Sliding clamp: follows DNA pol III to make sure it doesn’t fall off the strand

5. Clamp loader: Loads sliding clamp onto DNA strands

6. DNA ligase: binds okazaki fragments

7. Single-strand DNA binding proteins

Why does supercoiled DNA move faster through a gel?

The supercoiling makes the DNA radius smaller so it has an easier time moving through the gel.

What is BrdU? What is it used for?

A synthetic chemical compound that replaces thymidine during S phase and tags cells that are actively replicating.

What is Orc? Where would you find Orc in relation to chromatin?

The origin recognition complex made of 6 proteins. Can be found at the origin of replication at the beginning of S phase.

In animals, telomeres consist of TTAGGG repeats, while in plants, telomeres consist of TTTAGGG
repeats.
a) What does TERT do?
b) What does TERC do?

a) the protein component that has reverse transcriptase activity; uses TERC as a guide to extend the end of telomeres.

b) Is the RNA component and is the guide RNA used for extending the end of telomeres.

Transcriptional vs. Translational reporters

Transcriptional

Design: promoter region is fused directly upstream of the reporter gene, reporter has its own start site.

Function: tells you where and when the gene is transcribed.

Localization: resulting reporter gene typically stays in the cytoplasm.

Translational

Design: the regulatory region and coding sequence are fused in frame removing stop codons between.

Function: Reveals subcellular localization and real time trafficking of protein.

How is eukaryotic DNA packaged?

Double-stranded DNA wraps around histone octamers, which are then brought together by linker histones, making fibers (solenoid or zigzag pattern). These fibers form loops that make up the chromosome structure.

What do bacteria and archaae use to bind and compact DNA?

Nucleoid-associated proteins (NAPs).

Describe the process of centromere identification and kinetochore assembly.

• H3 and CenH3 nucleosomes are packaged so CenH3 are on the outside face of the chromosome

• Kinetochore proteins assemble on centromeric chromosomes

• Microtubules capture kinetochores

Heterochromatin vs. euchromatin

Heterochromatin

• tightly packed

• genetically inactive form of DNA that silences gene expression

Euchromatin

• loosely packed

• genetically active form of DNA that allows transcription

Why make polytene chromosomes?

• Massive gene amplification

• High-volume protein synthesis

• Cell growth

• Active transcription puffs

What are chromosome puffs?

Uncoiled regions on chromosomes that act as hotbeds for intensive RNA transcription.

Type I vs. Type II topoisomerases

Type 1 Topoisomerases

• Breaks only one strand of DNA helix

• Do not use ATP

• Can only relieve negative supercoiling

Type II Topoisomerases

• Breaks both strands of DNA helix

• Use ATP

• Can relieve both positive and negative supercoiling