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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