Principles of Genetic Technology (Cambridge (CIE) A Level Biology): Flashcards

Extracted directly from the DNA of a donor organism.

Synthesised from mRNA of the donor using reverse transcriptase.

Synthesised chemically from nucleotides.

Reverse transcriptase uses the mRNA as a template to make a complementary cDNA copy of the gene.

cDNA is DNA synthesised from an mRNA template by the enzyme reverse transcriptase.

Such cells contain many mRNA copies of that gene, giving an abundant, easy source to reverse transcribe into cDNA.

It can be synthesised chemically by joining nucleotides together in the correct order.

A gene can be made from an mRNA template using the enzyme reverse transcriptase.

They cut DNA at specific base sequences (recognition sites), often leaving short single-stranded sticky ends.

Sticky ends are short, unpaired single-stranded sections of DNA left when a restriction endonuclease makes a staggered cut.

Complementary sticky ends allow the gene and vector DNA to base pair and join together.

DNA ligase joins the sugar-phosphate backbones of two pieces of DNA by forming phosphodiester bonds, sealing the gene into the vector.

It synthesises a complementary DNA (cDNA) strand from an mRNA template.

It synthesises a complementary DNA strand from a template, e.g. converting single-stranded cDNA into double-stranded DNA.

So that both produce complementary sticky ends that can base pair with each other.

The enzyme that seals the gene into the vector by forming phosphodiester bonds is DNA ligase.

A vector is a vehicle used to carry the desired gene into the host cell, e.g. a plasmid or a virus.

A plasmid is a small, circular piece of DNA found in bacteria, separate from the main chromosome.

They are easily cut open and taken up by bacteria, can replicate independently, and often carry marker genes.

A plasmid that has had the desired gene inserted into it, forming recombinant DNA.

The plasmid is cut open with the same restriction endonuclease used on the gene.

DNA ligase then joins the gene into the plasmid via complementary sticky ends.

A small circular piece of bacterial DNA used to carry a gene into a host cell is a plasmid.

A promoter is a region of DNA, near the start of a gene, where RNA polymerase binds to begin transcription.

Without a promoter, RNA polymerase cannot bind, so the gene would not be transcribed and the protein not made.

For a transferred gene to be transcribed, RNA polymerase must bind to a region of DNA called the promoter.

A marker gene is a gene transferred alongside the desired gene, used to identify cells that have successfully taken up the desired gene.

Cells that have taken up and expressed the gene also express the marker, so they fluoresce (e.g. GFP glows green under UV light) and can be identified.

It can be seen directly (glows under UV light) without killing the cells, so successfully modified cells can be selected.

True.

Cells expressing the marker (e.g. fluorescing) are known to have taken up the desired gene.

Gene editing is a form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome.

Gene editing makes precise, targeted changes at specific sites in the organism's own genome, rather than transferring in a gene at a random location.

Insertion of DNA.

Deletion of DNA.

Replacement of DNA.

True.

It deliberately manipulates genetic material, changing DNA at specific target sites in the genome.

Making precise changes to DNA at specific sites in the genome is called gene editing.

Polymerase Chain Reaction — it amplifies (makes many copies of) a specific DNA fragment in vitro.

Denaturation (~95 °C): DNA strands separate as hydrogen bonds break.

Annealing (~55 °C): primers bind to the ends of the target sequence.

Extension (~72 °C): Taq polymerase builds new complementary strands.

Heating to ~95 °C breaks the hydrogen bonds, separating the double helix into two single strands.

Short single-stranded DNA sequences that anneal to the ends of the target sequence, providing a start point for DNA polymerase.

It adds complementary nucleotides to build new DNA strands from the primers.

Being from a thermophilic bacterium, it is heat-stable and is not denatured at high temperatures.

Each cycle doubles the number of DNA molecules (1 → 2 → 4 → 8 …).

The heat-stable enzyme that builds new DNA strands during PCR is Taq polymerase.

A technique that separates DNA fragments according to their length (size) using an electric field.

DNA is negatively charged (due to its phosphate groups), so it is attracted to the positive electrode (anode).

Smaller fragments move more easily through the pores of the gel, so they travel further; larger fragments move more slowly and stay nearer the wells.

DNA samples are loaded into wells in an agarose gel.

An electric current is applied.

Fragments separate by size as they move through the gel.

Fragments are stained or labelled to make the bands visible.

They are stained with a dye or labelled with fluorescent/radioactive markers and viewed, e.g. under UV light.

False.

Larger fragments move more slowly and travel a shorter distance; smaller fragments travel further.

In gel electrophoresis, DNA fragments are separated according to their length.

A slide carrying a grid of thousands of known single-stranded DNA probes at fixed positions, used to detect specific DNA or mRNA sequences.

Labelled (fluorescent) sample DNA/cDNA hybridises (base pairs) with complementary probes; spots that fluoresce show which sequences are present.

mRNA is converted to fluorescently labelled cDNA, which hybridises to probes; the spots that fluoresce reveal which genes are being transcribed (expressed).

They can compare genomes and detect the presence or absence of specific genes or alleles, e.g. comparing DNA from two individuals or species.

The labelled sample contained DNA/mRNA complementary to that probe, so it has hybridised — the sequence (or gene) is present or expressed.

In a microarray, labelled sample DNA binds to complementary probes by a process called hybridisation.

Bioinformatics is the use of computers and software to collect, store and analyse biological data, such as DNA and protein sequences.

Nucleotide sequences of genes and genomes, and the amino acid sequences and structures of proteins.

Scientists worldwide can access and share data instantly.

Sequences can be compared (e.g. between species or individuals) to study evolution and identify genes.

It can reveal evolutionary relationships, identify genes or mutations linked to disease, and find similar genes in other organisms.

The use of computers to store and analyse biological data such as DNA sequences is called bioinformatics.