Quick questions on PCR, gel electrophoresis, recombinant DNA, GMOs and CRISPR (QCE Biology Unit 4)

Purpose. Amplifies a specific DNA region from a tiny starting sample to billions of copies in a few hours.

Purpose. Separates DNA fragments by size.

Purpose. Combines DNA from different sources into a single molecule, usually to express a foreign gene in a host organism.

Definition. Organisms that carry a foreign gene introduced by recombinant DNA technology. The foreign gene is often called a transgene.

Origin. Adapted from a bacterial adaptive immune system that captures viral DNA fragments and uses them as guides to cut viral DNA on re-infection.

DNA sequencing. Sanger sequencing (chain-termination) for short reads; next-generation sequencing (Illumina, Oxford Nanopore) for whole genomes.

Amplifies a specific DNA region from a tiny starting sample to billions of copies in a few hours.

Template DNA, two primers (each 18 to 22 bases long, complementary to sequences flanking the target), the four dNTPs, a heat-stable DNA polymerase (Taq, from the hot-spring bacterium Thermus aquaticus), buffer with Mg2 plus ions, and a thermal cycler.

Forensic DNA profiling, paternity testing, prenatal genetic screening, diagnosis of bacterial and viral infections (including the SARS-CoV-2 RT-PCR test, where reverse transcriptase converts RNA to cDNA first), conservation genetics, ancient DNA studies.

DNA samples are loaded into wells in an agarose gel sitting in a buffered tank. An electric field is applied with the cathode (negative) at the loading end and the anode (positive) at the far end. DNA is uniformly negatively charged (because of the phosphate backbone), so it migrates toward the anode.

A DNA ladder (mixture of known-size fragments) is run alongside. After running, the gel is stained (ethidium bromide or a safer alternative such as SYBR Safe) and visualised under UV light. Band sizes are read off by comparison with the ladder.

The human insulin gene is inserted into E. coli, which transcribe and translate it to produce human insulin in industrial fermenters. This replaced pig and cow insulin in the 1980s.

Organisms that carry a foreign gene introduced by recombinant DNA technology. The foreign gene is often called a transgene.

Adapted from a bacterial adaptive immune system that captures viral DNA fragments and uses them as guides to cut viral DNA on re-infection.

Off-target edits, germline editing in humans (ethics), uneven global regulation.