3.8.4 Recombinant DNA technology

It performs transcription in 'reverse' (RNA to DNA).

Answer: It synthesises a single strand of complementary DNA (cDNA) from an mRNA template.

Fragments of DNA can be produced by several methods, including: conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase. This is an enzyme naturally found in retroviruses.

Think of them like puzzle pieces that only fit perfectly with a matching, complementary piece.

Answer: Sticky ends leave exposed, unpaired bases that can easily form hydrogen bonds with complementary sticky ends on a vector cut with the same enzyme.

Restriction enzymes cut DNA. If they cut in a staggered fashion, they leave 'sticky ends' (short, single-stranded overhangs). If the DNA fragment and the vector (e.g., plasmid) are cut with the same enzyme, their sticky ends will be complementary and can base-pair.

A gene cannot produce a protein if the cell doesn't know where to start and stop 'reading' it.

Answer: To provide binding sites for RNA polymerase to initiate and stop transcription of the inserted gene.

The addition of promoter and terminator regions to the fragments of DNA. A promoter is required for RNA polymerase and transcription factors to bind and begin transcription. A terminator tells RNA polymerase when to detach.

It acts as the final 'glue' to permanently seal the DNA strands together.

Answer: It forms phosphodiester bonds between the sugar-phosphate backbones of the DNA fragment and the vector.

The use of restriction endonucleases and ligases to insert fragments of DNA into vectors. After sticky ends pair via hydrogen bonds, DNA ligase catalyses the formation of strong phosphodiester bonds to permanently unite the backbones.

Transformation is a highly inefficient process; you need a way to 'spot' the few successful cells.

Answer: To identify which host cells have successfully taken up the recombinant plasmid.

The use of marker genes to detect genetically modified (GM) cells or organisms. Since only a small fraction of cells will take up the plasmid, the marker gene allows scientists to easily identify and isolate those successful transformants.

Heat provides kinetic energy to break weak bonds.

Answer: The hydrogen bonds between the complementary base pairs break, separating the double-stranded DNA into two single strands.

The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments. Heating to $95^{\circ}C$ separates the DNA strands. Cooling to $55^{\circ}C$ allows primers to anneal. Heating to $72^{\circ}C$ allows Taq polymerase to synthesize DNA.

It was originally discovered in bacteria living in hot springs.

Answer: It is thermostable and does not denature at the high temperatures ($95^{\circ}C$) required to separate the DNA strands.

The principles of the polymerase chain reaction (PCR). Standard human DNA polymerase would denature during the $95^{\circ}C$ heating phase. Taq polymerase is thermostable, so the cycle can be repeated continuously without adding new enzyme.

It 'searches' for a specific sequence by binding to it and lighting up.

Answer: A short, single-stranded piece of DNA that is radioactively or fluorescently labelled and is complementary to a specific target allele.

The use of labelled DNA probes and DNA hybridisation to locate specific alleles of genes. A probe is a single-stranded sequence that hybridises to its complementary target allele and is labelled for easy detection.