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Developing suitable methods for locating and isolating genes of interest isan important part of gene technology
There are three main methods for obtaining genes
• Synthesising the gene using an automated gene machine – this method canbe used if the amino sequence of the protein gene product is known; the
DNA sequence of the gene can be determined by working backwards usingthe genetic code
As most gene products are large proteins, this method isuseful mainly for smaller genes
• Shotgunning – this method involves isolating a gene from the entire genome; thetotal 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 ofhuman insulin
Isolating and Locating Genes Isolating and Locating Genes
Shotgunning – Isolating and locating the gene for Human Growth HormoneMost cells in the human body contain the entire human genome (total DNA) – exceptions
are gametes and red blood cellsMany copies of the genome are obtained from human white blood cells and each genome
is incubated with restriction enzyme
The restriction enzyme (Bam H1 in this case) ‘cuts’ the DNA at specific
restriction sitesrestriction sites
Numerous fragments of varying length are produced fromeach genome; one fragment from each genome contains the
human growth hormone gene
growth hormonegene
restriction fragments
TotalDNA
Isolating and Locating Genes Isolating and Locating Genes
The vector to be used in this example is the plasmid pBR322pBR322 is a manufacturedvector with genes coding
for resistance to theantibiotics ampicillin (amp)
and tetracycline (tet)
pBR322 has restrictionsites for a number of
restriction enzymes includingBam H1
pBR322 plasmids areincubated with the restriction
enzyme Bam H1 that ‘cuts’the plasmid in the region of
the tetracycline-resistance gene
restrictionsites
Isolating and Locating Genes Isolating and Locating Genes
Both the human DNA and the pBR322 plasmids have been ‘cut’ withthe SAME restriction enzyme; ‘cut’ plasmids and human DNA
fragments will therefore have the same ‘sticky ends’
Isolating and Locating Genes Isolating and Locating Genes
The three major products after recombination has occurred are:
Human DNA fragments thathave formed circular molecules
Re-sealed, non-recombinantplasmids that have not taken
up any foreign DNA
Recombinant Plasmids thathave taken up fragments
of the foreign, human DNA
Isolating and Locating Genes Isolating and Locating Genes
E. coli bacterial
cell
Uptake of DNA molecules
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 bacteriain the population of hostcells will fail to take up
any DNA molecules
Of those bacteria that do take up DNA
molecules, only a verysmall proportion will
contain the recombinant plasmidwith the desired gene
The task is to identify those bacterial host cells that have been transformed by recombinant plasmids containing the desired gene (growth hormone gene)
Bacteria containingcircular moleculesof human DNA donot form coloniesas they lack the
ampicillin resistance gene
The bacteria are transferred to nutrient agar platescontaining the antibiotic ampicillin
Bacteria containing any one of these plasmids will grow on the nutrient agaras these bacteria now possess a gene for resistance to ampicillin
The bacterial coloniesgrowing on this platecontain recombinantand non-recombinant
plasmids
Identifying the Required BacteriaIdentifying the Required Bacteria
non-recombinant
The bacteria growing on the ampicillin nutrient plates may contain eitherrecombinant or non-recombinant plasmids
The next task is todetermine which of
the bacterial coloniesgrowing on the ampicillin
plate have taken uprecombinant plasmids
as some of these bacteriacontain the desired gene
non-recombinant
Bacteria containing non-recombinant plasmids are resistant to both ampicillinand tetracycline; bacteria containing recombinant plasmids are resistant only
to ampicillin as their plasmids have human DNA fragments spliced intothe tetracycline-resistance gene
The bacteria growingon this ampicillin plateare now replica-platedonto plates containing
the antibiotic tetracycline
Bacteria containing therecombinant plasmidswill be unable to grow;
bacteria containingnon-recombinant plasmids
will continue to grow
Replica plating is a technique that allows molecular biologists to transfersamples 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
Replica Plating Tool
handle
sterilised felt orvelvet surface
The felt or velvet-coveredtool is pressed gently
onto the surface of thefirst agar plate containing
colonies of bacteria
Cells from each of thebacterial colonies stick to the velvet and can be transferred to thereplica plate in the
same positions relativeto one another The bacteria growing
on the ampicillin plate (i.e.all those containing plasmids)
are now replica plated onto a tetracycline plate
Replica PlatingReplica Plating
Tetracycline PlateAmpicillin Plate
sterile velvetsurface
pressed onto agarsurface of ampicillin
plate
samples of coloniesthat grow on ampicillin
are transferred to the velvet surface
colonies that grow onampicillin are replica-platedonto a tetracycline medium
Tetracycline plate on which only bacteria containing non-recombinant plasmids grow
non-recombinant
Ampicillin plate on which all bacteria containing plasmids grow
These colonies aremissing and show
where, on the ampicillin plate, bacterial colonies
containing recombinant
plasmids are growing
These coloniescontain
recombinantplasmids; thecolonies are
removed andgrown on a
new agar plate
Finding the Colonies of Bacteria with Recombinant Plasmids thatcontain the Desired Gene (Human Growth hormone gene)
The next task is tolocate 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
T
C
G
A
T
T
G
T
G
A
32P
T
32P
C
32P
G
32P
Nucleotides containing 32P are used to synthesise the gene probe
C
G
C
T
A
A
C
A
A Part of the‘sense strand’
of DNA containingthe nucleotide sequence
that forms the genefor human growth
hormone
A chain of radioactivenucleotides,
complementary to those of the gene sequence,
is synthesised
Radioactive geneprobe that will
hybridise (pair up)with the humangrowth hormone
gene
Making a Gene ProbeMaking a 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 placeda porous filter or membrane
Bacterial colonies from the master plate are replicaplated onto the surface of the porous filter
Bacterial colonies grow on the filterThe filter is removed and chemicallytreated in order to burst the bacterialcells and to make the released DNA
single-strandedSingle-stranded DNA is needed sothat the gene probe can bind to it
The filter is baked and now hassingle-stranded DNA molecules
bound to its structure in the positionsof the original bacterial colonies
Single-strandedDNA moleculesin the positionsof the original
bacterial colonies
Using the Gene ProbeUsing the Gene Probe
The filter is now incubatedin a solution containingradioactive gene probes
radioactivegene probesin solutionGene probe, with a sequence
of nucleotides that iscomplementary to that
of the DNA of the required gene (growth hormone gene),will hybridise (bind) to that
gene
The filter is removed from thesolution and excess probe is
washed away
The filter is then placed in contactwith an X-ray film for several days
Areas on the filter that containradioactive probe, and therefore
the desired gene, willblacken the film The blackened areas are compared
to the original master bacterial plates to reveal the colonies containing the required gene
The selected coloniesare isolated and cultured
to supply many copiesof the required gene
Using the Gene ProbeUsing the Gene Probe