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What are restriction enzymes?
Bacterial nucleases that generate double-strand DNA breaks.
What is the evolutionary purpose of restriction enzymes?
They evolved to protect bacteria from viruses.
Why don’t restriction enzymes cut the DNA of the species they originate from?
Their recognition sequence does not occur in their own genome
What are the types of cuts that restriction enzymes can produce?
Blunt ends, 5’ overhangs, and 3’ overhangs.
How does gel electrophoresis separate DNA fragments?
By size, using an electrical current.
Why does DNA migrate through the gel?
DNA carries a negative charge and moves toward the positive electrode.
How do DNA fragment sizes affect movement in gel electrophoresis?
Smaller fragments move faster than larger fragments.
What is the function of DNA ligase?
It uses ATP to join DNA fragments.
What natural role does DNA ligase play in cells?
It joins Okazaki fragments during DNA replication.
How is DNA ligase used in vitro?
It joins DNA fragments in cloning experiments.
What are plasmids?
Short, circular DNA molecules used by bacteria to share genes.
Why are DNA fragments often cloned into plasmids?
Linear DNA is difficult to propagate, while plasmids can replicate independently.
What allows plasmids to replicate in bacteria?
They contain their own replication origins.
How can restriction enzymes help in plasmid cloning?
They linearize plasmids for ligation with DNA fragments.
How are plasmids propagated in bacteria?
Through a process called transformation.
What is transformation?
When a bacterium takes up foreign DNA.
Why is transformation useful?
It allows bacteria to duplicate plasmids for research or genetic engineering.
What are genomic libraries?
Collections of DNA fragments from an entire genome stored in plasmids.
How are genomic libraries used in sequencing?
The genome is fragmented, cloned into plasmids, sequenced individually, and reassembled.
Why is direct cloning of eukaryotic genes difficult?
Eukaryotic genes contain introns, which complicate cloning
What enzyme creates DNA from RNA?
Reverse transcriptase.
How is reverse transcriptase used in cloning?
It converts mRNA into complementary DNA (cDNA) for cloning.
How is the poly-A tail of mRNA useful in cloning?
A poly-T primer can be used to initiate reverse transcription.
What is PCR used for?
Amplifying specific DNA sequences.
hat are the steps of PCR?
1) Denaturation (heat separates strands), 2) Annealing (primers bind), 3) Extension (DNA polymerase synthesizes new strands).
What enzyme makes PCR possible?
Heat-stable Taq polymerase.
How does PCR achieve exponential DNA amplification?
Each cycle doubles the amount of DNA.
What is RT-PCR used for?
Cloning cDNA from mRNA.
How does PCR aid molecular diagnostics?
It can detect minute amounts of DNA, such as in viral infections.
What is Real-Time PCR used for?
Quantifying the amount of starting DNA.
How does Real-Time PCR track DNA amplification?
By adding a fluorescent dye and measuring light intensity after each cycle
How can RT-PCR be used to measure gene expression?
It combines reverse transcription with real-time PCR to quantify mRNA levels.
What is in situ hybridization used for?
Detecting specific DNA or RNA sequences in cells and tissues.
How does in situ hybridization work?
Probes bind to complementary sequences and are chemically detected.
What technique combines in situ hybridization with karyotyping?
Fluorescence in situ hybridization (FISH).
How do reporter genes track gene expression?
They encode observable proteins (e.g., LacZ, GFP).
What is RNA interference (RNAi) used for?
Temporarily suppressing gene expression.
How can gene knockdowns be performed in nematode worms?
By feeding them bacteria that produce siRNA targeting specific mRNA.
How does targeted gene replacement work?
Homologous recombination integrates modified DNA into a genome.
What is Cas9?
A bacterial RNA-guided DNA endonuclease.
How does Cas9 target DNA?
It requires a guide RNA to find complementary DNA sequences.
How does Cas9 improve gene editing efficiency?
it introduces double-strand breaks, increasing homologous recombination efficiency.
How can Cas9 be modified to regulate genes?
By fusing it to regulatory proteins to turn genes on or off.
How are transgenic plants created?
By exposing plant cells to genetically modified Agrobacterium.
What are recombinant proteins?
Proteins produced by one organism using genes from another species.
Why can recombinant proteins be produced in any species?
The genetic code is universal.
What is an example of a recombinant protein used in medicine?
Insulin produced by E. coli or yeast for diabetes treatment.
