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Molecular Biology and Cloning Essentials
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Molecular Biology and Cloning Essentials
Molecular Biology and Cloning Essentials
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1
Question
What is the central dogma of molecular biology as described in the text?
Answer
DNA is transcribed to RNA, which is translated to protein.
2
Question
What distinguishes eukaryotic cells from prokaryotic cells in terms of organization and genome?
Answer
Eukaryotes are compartmentalized with organelles and more non-coding DNA; prokaryotes lack compartmentalization and have more coding DNA.
3
Question
Where does mRNA splicing occur and what is its consequence?
Answer
In the nucleus; introns are removed and exons exit for translation.
4
Question
Where do ribosomes function in eukaryotic cells according to the text, and what is implied about maturation?
Answer
Ribosomes are involved in protein synthesis with maturation occurring in the endoplasmic reticulum/Golgi.
5
Question
List reversible post-translational modifications mentioned.
Answer
Hydroxylation, phosphorylation, acetylation, methylation, AMP-ylation, ADP-ribosylation, glycosylation, prenylation, ubiquitilation, protein conjugation, redox.
6
Question
Name two irreversible post-translational modifications cited.
Answer
Proteolytic cleavage and amino acid modifications like deamidation/elimination.
7
Question
What factors influence protein charge and isoelectric point?
Answer
State of amino acid side chains and the pH of the environment.
8
Question
How is protein structure hierarchical according to the text?
Answer
Primary, secondary (alpha-helix/beta-sheet), tertiary, and quaternary structures.
9
Question
What determines the efficiency and location of protein expression in cells?
Answer
Promoter strength, regulatory elements, ribosome-binding sites, and targeting signals.
10
Question
What are the main cloning vectors and what is a key feature required for selection?
Answer
Plasmids, bacteriophages (λ, P1), yeast artificial chromosomes; antibiotic resistance marker.
11
Question
Why do restriction enzymes create sticky ends and how does this aid cloning?
Answer
They cut DNA to leave overhanging cohesive ends that facilitate ligation with inserts.
12
Question
What is transformation in bacterial cloning and what governs selection after transformation?
Answer
Uptake of plasmid DNA by bacteria; only transformed cells survive in antibiotic-containing medium.
13
Question
Compare universally: eukaryotes vs prokaryotes in gene data repositories for cloning.
Answer
Eukaryotes: cDNA libraries; Prokaryotes: genomic libraries.
14
Question
What approach is used to identify clones in a genomic library via DNA hybridization?
Answer
Hybridization with labeled complementary probes after DNA denaturation.
15
Question
What is the purpose of RACE in PCR-based cloning and what ends does it target?
Answer
Rapid Amplification of cDNA Ends to extend sequences at 5' or 3' ends.
16
Question
What is heterologous expression and why is it used?
Answer
Expression of a gene in a non-native host to increase production or study function.
17
Question
Name two prokaryotic expression hosts and two eukaryotic expression hosts listed.
Answer
Prokaryotes: E. coli, Lactobacillus; Eukaryotes: Yeasts (S. cerevisiae, Pichia pastoris), insect/mammalian cells.
18
Question
What is the difference between expression vectors and cloning vectors?
Answer
Cloning vectors replicate the gene fragment; expression vectors enable protein production.
19
Question
Explain the Lac and T7 systems and how IPTG functions in pET vectors.
Answer
Lac repressor controls transcription; IPTG binds Lac repressor, enabling transcription of T7 polymerase; T7 promoter drives the gene; pLys can shut down T7 with lysozyme.
20
Question
What is a shuttle vector and why is it advantageous?
Answer
A vector with two origins of replication (prokaryotic and eukaryotic) enabling cross-species manipulation.
21
Question
Describe the role of the Shine-Dalgarno sequence in prokaryotic translation.
Answer
It aligns mRNA on the ribosome by base-pairing with 16S rRNA to initiate translation.
22
Question
What is the significance of codon usage and codon degeneracy in cloning?
Answer
Codon preferences affect translation efficiency; degeneracy requires primers that accommodate variation.
23
Question
What is the purpose of using degenerate primers in PCR cloning?
Answer
To target conserved protein-coding regions when exact sequence is unknown.
24
Question
What are primers degatados (degenerate primers) and why are they used?
Answer
Degenerate primers contain mixed nucleotides to match conserved protein regions.
25
Question
What is the main goal of expressing proteins in yeast (S. cerevisiae) vs Pichia pastoris?
Answer
S. cerevisiae: robust genetics, slower growth; P. pastoris: stronger promoters, higher expression and secretion.
26
Question
What are epissomal vs integrative yeast vectors and their trade-offs?
Answer
Epissomal: plasmids, do not integrate; Integrative: integrate into genome, more stable but lower expression.
27
Question
Why is secretion of recombinant proteins advantageous in yeast expression systems?
Answer
Eases purification and can leverage secretory pathways.
28
Question
What is a major consideration when choosing expression host regarding post-translational modifications?
Answer
Requirement for glycosylation, disulfide bonds, and other modifications.
29
Question
What is the IPTG-induced system pET primarily dependent on?
Answer
T7 RNA polymerase activity driven by a T7 promoter.
30
Question
What are potential drawbacks of high promoter strength in expression systems?
Answer
More mRNA but potentially burdensome translation; misfolding or inclusion bodies possible.