Transcription, Translation, Replication

What is a nucleosome?

A loop of DNA and 8 histone proteins

What is chromatin?

A fiber of packed nucleosomes

What are chromosomes?

Chromatin that’s coiled together.

When are chromosomes formed?

Only when the cell is dividing

What is splicing?

The process of editing mRNA so that only the protein-coding regions (exons) are left and the introns are removed.

Where does splicing take place?

Inside the nucleus

What enzyme is responsible for unzipping the DNA?

Helicase

What strand is the leading strand? What strand is the lagging strand? Which one requires more complex copying?

1. Leading: 3’

2. Lagging: 5’

3. Complex copying: 5’

Which strand are Okazaki fragments associated with? What are their role in replication?

1. The 5’ lagging strand.

2. Helps the polymerase synthesize backwards

What carries amino acids into the ribosomes?

tRNA

On which type of RNA is the codon located? On which type of RNA is the anticodon located?

1. Codon: mRNA

2. Anticodon: tRNA

What does the small ribosomal subunit do? What does the large ribosomal subunit do?

1. Small: positions mRNA so it can be read by tRNA

2. Large: removes amino acid from tRNA and joins it to growing protein chain.

What are the three sites on a ribosome and what does each one do?

1. A-site: tRNA enters ribosome and is tested for codon-anticodon match

2. P-site: amino acid is added to chain if there’s a match

3. E-site: tRNA is ejected and recycled

What is the protein’s shape determined by?

The order of amino acids

What kind of chromosomes are affected by translocations?

Non-homologous chromosomes only (during mitosis)

What are four examples of point mutations?

1. Substitution

2. Transition

3. Transversion

4. Mispairing

What is a transition?

A swap between either two pyrimidines or two purines

What is a transversion?

Swap for a pyrimidine from purine

What is a mispairing?

When A-C or G-T

What are five applications of PCR?

1. DNA sequencing

2. DNA fingerprinting

3. Forensics

4. Detection of microorganisms

5. Diagnosis of hereditary disease

What are the four steps of the PCR process?

1. Initialization

2. Denaturation

3. Annealing

4. Extension

What is the purpose of the initialization phase?

1. Activates DNA polymerase and denatures contaminants

2. Facilitates cell lysis to release DNA and denature cellular proteins

What temperature is needed for initialization and how long does it take?

1. 94-96 degrees Celsius

2. 2-10 minutes

What happens in denaturation?

Hydrogen bonds between DNA strands are broken

What is the temperature associated with denaturation and how long does it take?

1. 94-98 degrees Celsius

2. 20-30 seconds

What happens during the annealing phase?

Primers bind to sequence on DNA to begin replication

What temperature is associated with annealing and how long does it take?

1. 50-65 degrees Celsius

2. 20-40 seconds

What happens during the extension phase?

DNA polymerase adds dNTP’s to synthesize new strand in the 5’ to 3’ direction.

What kind of DNA polymerase is used and why?

Taq polymerase, because it’s heat-labile

What temperature is associated with the extension phase?

72-78 degrees Celsius

What temperature are PCR samples stored at?

4-75 degrees Celsius

What technology allows PCR results to be visualized?

Gel electrophoresis that separates proteins based on size

What five factors influence how PCR is performed?

1. Composition of DNA template

2. DNA polymerase choice

3. Buffer components

4. Primer design

5. Additives and inhibitors

How does EDTA affect PCR?

It inhibits PCR

What are four types of PCR controls?

1. Positive

2. Negative

3. No Template Control

4. Internal Positive Control

What is the difference between a negative control and a no template control?

A negative control lacks the target sequence while the no template control lacks nucleotides.

Which control identifies contamination?

No template control

What does the internal positive control test for and what was used as the IPC in this rotation?

1. Tests polymerase function, mastermix components, cycling conditions, and detector system

2. Albumin

What does DNA topoisomerase do?

Create dsDNA breaks

What keeps the helix open and prevents it from zipping back up?

DNA binding proteins

What enzyme does Helicase activate, and what does this enzyme do?

1. Primase

2. Synthesizes a short RNA primer

What does DNA polymerase do?

Adds dNTP’s to the primer

What does DNA Ligase do?

Fuses okazaki fragments to each other via phosphodiester bonds

What happens when a DNA switches direction?

It begins proofreading

What does DNA polymerase g do?

Helps replicate and proofread mitochondrial DNA

What does the telomerase do?

Contains RNA template to extend telomere on lagging strand with 3’ overhang