Genomes and
gene technologies
By Daniella Di-Fonzo…read more

Specification
· Candidates should be able to:
· (a) outline the steps involved in sequencing the genome of an organism;
· (b) outline how gene sequencing allows for genome-wide comparisons between individuals and between species
(HSW7b);
· (c) define the term recombinant DNA;
· (d) explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of
genes, in order to place them in another organism (often of a different species) such that the receiving organism
expresses the gene product (HSW6a);
· (e) describe how sections of DNA containing a desired gene can be extracted from a donor organism using
restriction enzymes;
· (f) outline how DNA fragments can be separated by size using electrophoresis (HSW3);
· (g) describe how DNA probes can be used to identify fragments containing specific sequences;
· (h) outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments;
· (i) explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase;…read more

(j) state other vectors into which fragments of DNA may be incorporated;
· (k) explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism
that can express a desired gene product;
· (l) describe the advantage to microorganisms of the capacity to take up plasmid DNA from the
environment;
· (m) outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a
recombinant plasmid;
· (n) outline the process involved in the genetic engineering of bacteria to produce human insulin;
· (o) outline the process involved in the genetic engineering of `Golden RiceTM' (HSW6a);
· (p) outline how animals can be genetically engineered for xenotransplantation (HSW6a, 6b);
· (q) explain the term gene therapy;
· (r) explain the differences between somatic cell gene therapy and germ line cell gene therapy;
· (s) discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants
and microorganisms (HSW4, 6a, 6b, 7c).…read more

Sequencing a genome
· Genome can mean all genes of an individual organism or
all genes in a population of organisms.
· Gene technologies used to study genes and their functions
include:
· Polymerase chain reaction (PCR)
· Cutting out DNA fragments using restriction
enzymes
· Gel electrophoresis
· DNA probes…read more

Sequencing a Genome
If a whole genome needs sequencing: use PCR to make multiple copies of all the DNA.
Squeeze through a tiny hole under high pressure. Cuts DNA into 2000- 10000 base pairs.
These can be shortened to make it easier to sequence them.
Make multiple labelled copies of these lengths of DNA. They are mixed with a primer (up to 20
base pairs of DNA, complimentary to the start of the DNA fragment you want), free
nucleotides, DNA polymerase (to create new DNA strands) and nucleotides labelled with
fluorescent dye. Each base has it's own colour and each labelled nucleotide with stop the
chain from growing.
The DNA polymerase attaches free nucleotides to the lengths of DNA. Most of the copies wont
be complete due to the labelled nucleotides. This means lots of different length chains.
This is then separated using electrophoresis so the DNA is drawn towards the positive end of a
capillary tube.
A computer records the colours as they pass, the shorter the chain, the faster it travels, so if
there are enough fragments, the computer will be able to work out the sequence of bases in
that particular length of DNA.
This is then done for every piece of DNA and then a computer program works out the overall
sequence of nucleotides in the whole genome.…read more

PCR
PCR can be used to make millions of copies of a DNA fragment in just
a few hours.
1. DNA is denatured by heating to 95°C, to break
hydrogen bonds of the double helix of DNA, so the
base pairs are exposed.
2. The mixture is then cooled to between 50°C - 65°C so
the primer (up to 20 pieces of DNA complimentary to
the start of the DNA fragment you want) can bind
(anneal) to the strands.
3. The mixture is then heated to 72°C so DNA
polymerase can DNA strand.…read more