DNA cloning/ recombinant DNA technology

Why do scientists clone genes or DNA fragments?

To study gene function, express proteins, or create genetically modified organisms

What is a vector in molecular cloning?

A DNA molecule used to carry foreign DNA into a host cell

What are the main steps of gene cloning?

Isolate DNA, ligate into vector, transform cells, select successful clones

What is transformation?

Introduction of recombinant DNA into a host cell

What is selection in cloning?

Identifying cells that have taken up the correct recombinant vector

What are restriction endonucleases?

Enzymes that cut DNA at specific palindromic sequences

Where do restriction enzymes originate from?

Bacterial immune defense systems

Why must a restriction enzyme cut a plasmid only once?

To linearise the plasmid without fragmenting it

Why must the same restriction enzyme cut both insert and vector?

To create compatible DNA ends for ligation

What are sticky ends?

Single-stranded overhangs that facilitate DNA ligation

What are blunt ends?

DNA ends without overhangs that ligate less efficiently

What is ligation?

Joining DNA fragments together using DNA ligase

Why are sticky ends preferred over blunt ends?

They increase efficiency and specificity of ligation

What is PCR?

A method to amplify a specific DNA sequence in vitro

What are primers in PCR?

Short DNA sequences that define the region to be amplified

Why are two primers required in PCR?

One binds each DNA strand to enable replication

What is the role of Taq polymerase?

Heat-stable DNA polymerase that synthesizes DNA during PCR

Why is Taq polymerase heat stable?

It originates from thermophilic bacteria

What is denaturation in PCR?

Heating DNA to separate the two strands

What is annealing in PCR?

Primers binding to complementary DNA sequences

What is extension in PCR?

DNA synthesis by polymerase from the primers

What is RT-PCR?

PCR performed on cDNA made from mRNA

Why does cDNA lack introns?

Introns are removed during mRNA processing

What enzyme synthesises cDNA?

Reverse transcriptase

Why use RT-PCR for gene expression studies?

It amplifies only expressed genes

How can restriction sites be added to PCR products?

By including them in primer sequences

What is Gibson cloning?

A cloning method that joins DNA fragments without restriction enzymes

What enzyme creates sticky ends in Gibson cloning?

5’ exonuclease

What is the advantage of Gibson cloning?

Seamless insertion without restriction site constraints

Why is cloning necessary for CRISPR editing?

To deliver Cas9 and guide RNA into cells

What is ADE2 gene editing used to demonstrate?

Functional gene editing in yeast