Gentechnologie: Nucleic Acids, Replication, and Genetic Engineering

Practice flashcards covering DNA structure, replication, transcription, translation, and genetic engineering technologies including PCR, cloning, and CRISPR-Cas9 based on lecture notes.

What are the three main components of a nucleotide?

A phosphate group, a sugar (deoxyribose in DNA or ribose in RNA), and a nitrogenous base.

Which nitrogenous bases are classified as purines and which are pyrimidines?

Purines (larger structure with two fused rings) are Adenine and Guanine. Pyrimidines (smaller structure with a single ring) are Cytosine, Thymine, and Uracil (found in RNA).

What is the hyperchromic effect?

The phenomenon where single-stranded DNA (ssDNA) absorbs more UV light at $\sim 260\,\text{nm}$ than double-stranded DNA (dsDNA) because the bases are exposed rather than stacked and hidden.

Why does Poly(GC) DNA melt at a higher temperature ($Tm$) than Poly(AT) DNA?

G-C pairs have $3$ hydrogen bonds, while A-T pairs have only $2$ hydrogen bonds. More bonds make the DNA stronger and harder to separate.

What is the function of Topoisomerase I?

An enzyme responsible for adding and removing supercoils by breaking, unwinding, or overwinding DNA strands.

Distinguish between exonucleases and endonucleases.

Exonucleases release nucleotide residues from one end ($3' \rightarrow 5'$ or $5' \rightarrow 3'$) of a polynucleotide chain, whereas endonucleases cut at sites within the chain.

How do host cells protect their own DNA from restriction endonucleases?

By covalent modification of the bases (most commonly methylation of adenine or cytosine) at the potential binding site.

What characterizes 'semi-conservative' DNA replication?

Each of the two resulting identical DNA molecules contains one original template strand and one newly synthesized complementary strand.

Why does DNA polymerase III require a primer?

It cannot start synthesis from scratch; it needs a short starting piece of RNA (made by primase) to provide a $-OH$ group for extension.

What is the role of DNA polymerase I in joining Okazaki fragments?

It performs nick translation by extending the Okazaki fragment while its $5' \rightarrow 3'$ exonuclease activity removes the RNA primer.

What are the two repair pathways for double-strand breaks (DSB) created by Cas9?

Non-homologous end joining (NHEJ), which is fast but error-prone (leading to gene knockout), and Homology-directed repair (HDR), which is precise and uses a template for gene correction or insertion.

Which RNA polymerases are responsible for making rRNA, mRNA, and tRNA in eukaryotes?

RNA polymerase I makes rRNA, RNA polymerase II makes mRNA, and RNA polymerase III makes tRNA.

What is the difference between Rho-dependent and Rho-independent transcription termination?

Rho-dependent termination requires the Rho protein to unwind the RNA-DNA hybrid, while Rho-independent (spontaneous) termination relies on an inverted repeat forming a hairpin structure followed by a stretch of Uracil bases.

What occurs during RNA splicing in eukaryotes?

Introns (non-coding regions) are removed and exons (coding regions) are joined together to form mature mRNA.

Explain 'diauxic growth' in the context of E. coli and the Lac operon.

It is the two-phase growth pattern where bacteria first prefer glucose; once glucose is exhausted, a lag phase occurs to activate lactose metabolism genes before growth resumes using lactose.

What is the 'wobble position' in translation?

It is the atypical base pairing at the third position of a codon, allowing a single tRNA to recognize multiple codons for the same amino acid.

What are the standard temperature ranges and functions of the three PCR steps?

1. Initial Denaturation (high heat to separate strands), 2. Primer Annealing (typically $50-60\,^{\circ}\text{C}$ for primer binding), and 3. Primer Extension (polymerase synthesizes new DNA).

What is the difference between SYBR Green and probes in qPCR?

Dyes like SYBR Green bind to all double-stranded DNA universally, whereas probes are sequence-specific and only emit fluorescence when the target is amplified.

What are the two main methods for transformation (DNA uptake) in the lab?

Heat shock (using chemically competent cells at $42\,^{\circ}\text{C}$) and electroporation (using high-voltage electric pulses).

Compare Gibson Assembly and SLiCE (Seamless Ligation Cloning Extract).

Gibson Assembly uses a specific mix of purified enzymes (exonuclease, polymerase, ligase), while SLiCE uses a crude bacterial cell extract containing natural recombination enzymes, making it a cheaper alternative.