Molecular Biology Flashcards

Flashcards from Molecular Biology Lecture Notes

Explain the basis of Y2H (Yeast Two-Hybrid) and the roles of Gal4 domains.

Gal4 transcription has two domains: a DNA binding domain (BD) and a transcription activity domain (AD). These domains do not need to be in the same protein but must be in the same protein complex.

What do pGBD and pGAD plasmids code for in Yeast Two-Hybrid assays?

pGBD plasmids code for a fusion protein of Gal4-BD and the protein of interest, containing the TRP1 gene for selection. pGAD plasmids code for a fusion protein with Gal4-AD and protein of interest, containing the LEU2 gene for selection.

Describe 14-3-3 proteins and their effects on binding.

14-3-3 proteins are conserved dimers involved in many cellular processes, such as phosphorylation, interaction with other proteins, localization, and degradation.

How do Type II restriction enzymes cut DNA?

Type II restriction enzymes cut DNA next to their recognition site, creating 5’ sticky ends, 3’ sticky ends, or blunt ends.

What is the purpose of alkaline phosphatase treatment in cloning?

Alkaline phosphatase treatment removes the 5’-phosphate from the vector, preventing self-ligation and facilitating insert uptake.

What is the optimal ratio of vector to insert in a ligation reaction?

Optimal molar ratio is vector:insert = 1:3

Explain the principle behind blue-white screening.

In blue-white screening, active β-galactosidase tetramers form blue colonies. Cloning into the MCS within the α-peptide disrupts complementation, resulting in white colonies.

How do you confirm the presence and size of a fragment after plasmid isolation from E. coli?

To determine if a fragment is present and of the correct size, choose restriction enzymes that were used to cut the vector and fragment.

How do you determine the orientation of an insert in a vector?

To determine the orientation of a fragment, choose an enzyme that cleaves asymmetrically in the insert and vector.

What factors affect DNA migration during gel electrophoresis, and how is DNA visualized?

Gel electrophoresis separates DNA based on charge, size, shape, and voltage. Visualization uses fluorescent dyes and UV light.

What causes supercoiling in plasmids, and how does it affect gel migration?

Supercoiling is caused by DNA topoisomerases in bacteria. Supercoiled plasmids migrate differently than linear plasmids during gel electrophoresis.

Describe the Sanger sequencing method.

Sanger sequencing involves primer binding, DNA polymerase extension with dNTPs and ddNTPs, separation by length, and tag detection to determine DNA sequence.

What happens during the annealing stage of PCR?

During the annealing stage of PCR, primers bind to the DNA at 55-65℃, marking the start sites for DNA polymerase.

Describe the elongation phase of transcription.

In transcription, RNA polymerase II follows the template DNA strand from 3’ to 5’, synthesizing an RNA molecule that’s a copy of the coding strand.

What is the role of tRNA in translation?

tRNA brings the correct amino acid to the peptide chain and binds to mRNA with its anticodon during translation, which takes place in ribosomes.

Explain the role of signal peptides in protein localization.

A signal peptide, usually at the N-terminus, facilitates transport to membranes or organelles. The Signal Recognition Particle (SRP) recognizes the ER-signal peptide and binds to a receptor at the ER membrane pore.

What is the purpose of Southern and Western blotting?

Southern blotting is used to detect a specific DNA sequence in a sample, while Western blotting is used to detect a specific protein using antibodies.

Describe random primed DNA synthesis for creating labeled probes.

Random primed DNA synthesis involves denaturing a DNA fragment, annealing random hexamers, adding labeled dNTPs, and using Klenow fragment or DNA polymerase to build radioactive dNTPs.

How is the first strand of cDNA synthesized?

In cDNA synthesis, oligo dT DNA primers hybridize with mRNA poly-A tails, and reverse transcriptase extends the oligo dT primer.

How is SYBR Green used in qPCR?

In qPCR, SYBR Green's fluorescence, which increases upon binding to dsDNA, is measured after each elongation.

Describe the steps involved in RNA-sequencing (RNA-Seq).

RNA-sequencing involves isolating and fragmenting mRNA, creating cDNA, sequencing, and mapping to a reference genome to determine gene expression levels.

Describe site directed mutagenesis.

Site-directed mutagenesis: design primer with mutation, then DNA polymerase synthesizes the complement and ligase seals the gaps, followed by transformation into E.coli.

Describe Agrobacterium-mediated plant transformation.

In Agrobacterium-mediated plant transformation, the T-DNA integrates into the plant genome via NHEJ, potentially disrupting genes at the insertion site.

How does gene editing occur using CRISPR/Cas?

CRISPR/Cas: Cas9 protein makes a double-strand break (DSB) at the target site guided by sgRNA, and the repair is done

What is genetic complementation?

In genetic complementation, adding a wild-type gene to a mutant organism can restore function.