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Isolating and Locating Genes
Developing suitable methods for locating and isolating genes of interest is
an important part of gene technology
There are three main methods for obtaining genes
• Synthesising the gene using an automated gene machine – this method can
be used if the amino sequence of the protein gene product is known; the
DNA sequence of the gene can be determined by working backwards using
the genetic code
As most gene products are large proteins, this method is
useful mainly for smaller genes
• Shotgunning – this method involves isolating a gene from the entire genome; the
total DNA of the genome is ‘cut’ into fragments with restriction enzymes and the
fragment containing the desired gene is identified
• Using the enzyme Reverse Transcriptase – this method involves isolating
messenger RNA molecules from cells that manufacture a specific protein and
then using the enzyme Reverse Transcriptase to make a DNA copy (cDNA) of
the messenger RNA; this approach has been used for the manufacture of
human insulin
Isolating and Locating Genes
Shotgunning – Isolating and locating the gene for Human Growth Hormone
Most cells in the human body contain the entire human genome (total DNA) – exceptions
are gametes and red blood cells
Many copies of the genome are obtained from human white blood cells and each genome
is incubated with restriction enzyme
Total
DNA
The restriction enzyme (Bam H1 in this
case) ‘cuts’ the DNA at specific
restriction sites
restriction sites
Numerous fragments of varying length are produced from
each genome; one fragment from each genome contains the
human growth hormone gene
restriction fragments
growth hormone
gene
Isolating and Locating Genes
The vector to be used in this example is the plasmid pBR322
pBR322 is a manufactured
vector with genes coding
for resistance to the
antibiotics ampicillin (amp)
and tetracycline (tet)
restriction
sites
pBR322 has restriction
sites for a number of
restriction enzymes including
Bam H1
pBR322 plasmids are
incubated with the restriction
enzyme Bam H1 that ‘cuts’
the plasmid in the region of
the tetracycline-resistance gene
Isolating and Locating Genes
Both the human DNA and the pBR322 plasmids have been ‘cut’ with
the SAME restriction enzyme; ‘cut’ plasmids and human DNA
fragments will therefore have the same ‘sticky ends’
Isolating and Locating Genes
The three major products after recombination has occurred are:
Human DNA fragments that
have formed circular molecules
Re-sealed, non-recombinant
plasmids that have not taken
up any foreign DNA
Recombinant Plasmids that
have taken up fragments
of the foreign, human DNA
The various DNA molecules are now mixed with a population of the host cells – the
bacterium E.coli (a strain that is NOT resistant to either ampicillin or tetracycline)
Some of the bacteria
in the population of host
cells will fail to take up
any DNA molecules
Uptake of DNA molecules
Of those bacteria
that do take up DNA
molecules, only a very
small proportion will
contain the
recombinant plasmid
with the desired gene
E. coli
bacterial
cell
The task is to identify those bacterial host cells that have been transformed by
recombinant plasmids containing the desired gene (growth hormone gene)
Identifying the Required Bacteria
The bacteria are transferred to nutrient agar plates
containing the antibiotic ampicillin
Bacteria containing any one of these plasmids will grow on the nutrient agar
as these bacteria now possess a gene for resistance to ampicillin
Bacteria containing
circular molecules
of human DNA do
not form colonies
as they lack the
ampicillin
resistance gene
The bacterial colonies
growing on this plate
contain recombinant
and non-recombinant
plasmids
The bacteria growing on the ampicillin nutrient plates may contain either
recombinant or non-recombinant plasmids
nonrecombinant
The next task is to
determine which of
the bacterial colonies
growing on the ampicillin
plate have taken up
recombinant plasmids
as some of these bacteria
contain the desired gene
Bacteria containing non-recombinant plasmids are resistant to both ampicillin
and tetracycline; bacteria containing recombinant plasmids are resistant only
to ampicillin as their plasmids have human DNA fragments spliced into
the tetracycline-resistance gene
nonrecombinant
The bacteria growing
on this ampicillin plate
are now replica-plated
onto plates containing
the antibiotic
tetracycline
Bacteria containing the
recombinant plasmids
will be unable to grow;
bacteria containing
non-recombinant plasmids
will continue to grow
Replica Plating
Replica plating is a technique that allows molecular biologists to transfer
samples of bacterial colonies from one nutrient agar plate to another
Using this method, duplicate bacterial samples can be grown on a second agar
plate in exactly the same position that they were growing on the first, master plate
The felt or velvet-covered
tool is pressed gently
onto the surface of the
first agar plate containing
colonies of bacteria
handle
Cells from each of the
bacterial colonies stick
to the velvet and can
be transferred to the
replica plate in the
same positions relative
to one another
sterilised felt or
velvet surface
Replica Plating Tool
The bacteria growing
on the ampicillin plate (i.e.
all those containing plasmids)
are now replica plated
onto a tetracycline plate
sterile velvet
surface
samples of colonies
that grow on ampicillin
are transferred to the velvet surface
pressed onto agar
surface of ampicillin
plate
Ampicillin Plate
colonies that grow on
ampicillin are replica-plated
onto a tetracycline medium
Tetracycline Plate
nonrecombinant
These colonies are
missing and show
where, on the
ampicillin plate,
bacterial colonies
containing
recombinant
plasmids
are growing
These colonies
contain
recombinant
plasmids; the
colonies are
removed and
grown on a
new agar plate
Ampicillin plate on which all bacteria
containing plasmids grow
Tetracycline plate on which only bacteria
containing non-recombinant plasmids grow
Finding the Colonies of Bacteria with Recombinant Plasmids that
contain the Desired Gene (Human Growth hormone gene)
The next task is to
locate bacterial
colonies containing
recombinant
plasmids with
the desired gene
Genetic engineers use Gene Probes to locate specific genes
A gene probe is a relatively short, single-stranded DNA molecule consisting
of around 15 to 20 nucleotides
Provided that at least part the base sequence of a particular gene is known,
it is possible to synthesise a sequence of nucleotides that is complementary
to that of the gene
Nucleotides containing 32P (radioactive phosphorus) are used to synthesise
the gene probe which in turn becomes a radioactive molecule
Making a Gene Probe
Nucleotides containing 32P are used to synthesise the gene probe
32
32
P
A
A
G
C
T
A
A
C
A
C
32
P
Part of the
‘sense strand’
of DNA containing
the nucleotide sequence
that forms the gene
for human growth
hormone
32
P
T
P
C
T
C
G
A
A chain of radioactive
nucleotides,
complementary to those
of the gene sequence,
is synthesised
T
T
G
T
G
Radioactive gene
probe that will
hybridise (pair up)
with the human
growth hormone
gene
G
Using the Gene Probe
Colonies of bacteria containing recombinant plasmids,
some of which will contain the required gene (growth
hormone gene)
Master Plate
A new agar plate is prepared onto which is placed
a porous filter or membrane
Bacterial colonies from the master plate are replica
plated onto the surface of the porous filter
Single-stranded
Bacterial colonies grow on the filter
DNA molecules
The filter is removed and chemically in the positions
treated in order to burst the bacterial of the original
cells and to make the released DNA bacterial colonies
single-stranded
Single-stranded DNA is needed so
that the gene probe can bind to it
The filter is baked and now has
single-stranded DNA molecules
bound to its structure in the positions
of the original bacterial colonies
Using the Gene Probe
radioactive
gene probes
in solution
The filter is now incubated
in a solution containing
radioactive gene probes
Gene probe, with a sequence
of nucleotides that is
complementary to that
of the DNA of the required
gene (growth hormone gene),
will hybridise (bind) to that
gene
The filter is removed from the
solution and excess probe is
washed away
The filter is then placed in contact
with an X-ray film for several days
The selected colonies
Areas on the filter that contain
are isolated and cultured
radioactive probe, and therefore
to supply many copies
the desired gene, will
of the required gene
blacken the film
The blackened areas are compared
to the original master bacterial plates
to reveal the colonies containing the required gene