Recombinant DNA Technology and Central Dogma — Flashcards

Flashcards covering key concepts from lecture notes on recombinant DNA technology, central dogma, cloning, PCR, libraries, metagenomics, genome editing, transgenesis, and CRISPR.

What is the Central Dogma and its flow of information?

Flow is DNA (genotype) -> RNA (transcript) -> protein (phenotype); one-way path, with an exception via reverse transcription in retroviruses.

Which enzyme copies DNA during replication and what does replication require?

DNA polymerase; requires a primer to start synthesis and produce identical DNA molecules.

What is transcription and which enzyme carries it out?

RNA polymerase synthesizes RNA using DNA as a template; produces mRNA that carries the code to the cytoplasm (T in DNA is replaced by U in RNA).

What is translation and what machinery carries it out?

Ribosome translates mRNA into protein in the cytoplasm.

What are the key regulatory domains of a gene?

Promoter, activator, silencer, terminator; the open reading frame (ORF) contains exons and introns (introns are present in eukaryotes, not typically in bacteria).

What is the difference between exons and introns and which organisms have introns?

Exons code for the protein; introns are non-coding regions removed by splicing; introns are present in eukaryotes, not in most bacteria.

What is reverse transcription?

The process by which RNA-dependent DNA polymerase (reverse transcriptase) makes DNA from an RNA template; an exception to the one-way central dogma.

What is the role of a promoter in transcription?

Promoter binds RNA polymerase and determines where transcription starts; without it, basal expression would be absent.

What are transcription start sites and the start/stop elements in transcription and translation?

Transcription start site marks where RNA synthesis begins; start codon is ATG; stop codon signals termination; transcription terminator; there are 5' and 3' untranslated regions (UTRs).

What are untranslated regions (UTRs)?

Flanking regions of mRNA that are not translated into protein; influence translation efficiency and mRNA stability.

What do restriction enzymes do?

Cut DNA at specific restriction sites, generating restriction fragments; used to create sticky or blunt ends for cloning.

What are sticky ends and blunt ends in restriction digestion?

Sticky ends are staggered cuts with overhangs that base-pair with complementary ends; blunt ends are straight cuts with no overhangs; ligation efficiency varies.

What is EcoRI and why is it important?

A commonly used restriction enzyme from E. coli that recognizes a palindrome and creates sticky ends; widely used in cloning.

What is DNA ligase?

Enzyme that seals phosphodiester bonds to join DNA fragments after restriction digestion; forms a continuous backbone.

What is a plasmid and why is it used as a cloning vector?

Small circular DNA that replicates independently; carries an origin of replication, promoter, multiple cloning site, and selectable marker to propagate inserted genes in bacteria.

How does LacZ/X-gal blue-white screening work?

LacZ encodes beta-galactosidase; insertion into LacZ disrupts enzyme function; colonies with intact LacZ appear blue on X-gal plates; white colonies indicate inserts (recombinant plasmids).

What are the three possible outcomes when inserting a fragment with sticky ends into a vector?

Insert ligates in the correct orientation, in the reverse orientation, or the vector closes without an insert (empty vector).

What is transformation and what methods are used to introduce plasmids into bacteria?

Process of making bacteria take up plasmid DNA; methods include chemical transformation (salt and heat shock) and electroporation.

Why is antibiotic selection used in cloning?

Plasmids carry antibiotic resistance genes; only bacteria that have taken up the plasmid survive on antibiotic-containing media.

How can lacZ-based screening distinguish recombinant plasmids?

Insertion disrupts LacZ activity; white colonies indicate recombinant plasmids, blue colonies indicate non-recombinant plasmids.

What is a genomic DNA library?

A collection of plasmids each carrying a fragment of the organism's entire genome; used to locate or isolate genes.

What is a cDNA library and why is it useful?

A library of complementary DNA generated from mRNA; lacks introns and reflects expressed genes; can be expressed in prokaryotes.

What is a metagenomic library?

A library created from environmental DNA to access genes from organisms that cannot be cultured in the lab.

What is PCR and what are its main components?

In vitro DNA amplification; requires DNA template, primers, DNA polymerase, dNTPs, and buffer; performed in a thermal cycler.

What are the typical steps in a PCR cycle?

Denaturation (~95°C) to separate strands, annealing (primer-dependent temperature) to bind primers, extension (~72°C) to synthesize DNA; repeated for ~30 cycles.

What is a thermocycler?

Instrument that precisely cycles temperatures for PCR to enable DNA amplification.

How do you produce a genomic DNA library? (Steps summarized)

Isolate organism's DNA, fragment with restriction enzymes, ligate fragments into plasmids, transform bacteria, select white colonies to indicate inserts.

How can short oligonucleotides be assembled into longer DNA sequences?

Overlap-based assembly: design overlapping oligos, anneal, extend to fill gaps with PCR, denature and reassemble; repeat to build longer sequences (up to about 1000 bases).

What is codon optimization and why is it used?

Modify gene sequence to match the host's preferred codon usage to improve protein expression and yield.

What is the difference between constitutive and inducible promoters?

Constitutive promoters drive constant expression; inducible promoters require a stimulus to activate transcription.

Give an example of overexpression affecting phenotype.

Overexpression of enzymes (e.g., PEP carboxylase) can alter metabolism and phenotype (e.g., obesity in mice) depending on regulation.

What is antisense-mediated gene silencing?

Antisense RNA binds complementary mRNA, blocking translation or triggering degradation to reduce gene expression.

What is RNA interference (RNAi) and how does it work?

Double-stranded RNA is processed into siRNA/miRNA; guides RISC to complementary mRNA to degrade it, silencing gene expression; may involve stem-loop structures in some systems.

What is cisgenesis?

Using DNA from the same species (or a sexually compatible donor) to modify an organism; no foreign DNA introduced.

What is CRISPR-Cas9 and how does it edit genomes?

Cas9 nuclease guided by a gRNA creates a targeted double-strand break; repair by non-homologous end joining or homology-directed repair yields insertions/deletions or precise edits; used for genome editing in plants, animals, and humans (e.g., malaria-resistant mosquitoes).

What is the Minimal Genome Project?

Determining the smallest set of genes required for survival; Mycoplasma genitalium had ~415 genes, reduced to ~372; minimal genome synthesized and transplanted into recipient cells to create a minimal organism (Mycoplasma laboratorium).

What is transgenesis?

Introduction of artificial genetic constructs into a genome to alter expression; can achieve overexpression or gene silencing using promoters (constitutive or inducible) and terminators; orientation affects expression.

What are the two main methods of gene silencing described (antisense and RNAi)?

Antisense RNA binds to target mRNA to block translation or trigger degradation; RNAi uses dsRNA processed into siRNA/miRNA to guide degradation of target mRNA.