SSER Ltd Isolating and Locating Genes Developing suitable

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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 (c. DNA) 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 H 1 in this case) ‘cuts’ the DNA at specific 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 p. BR 322 is a manufactured vector with genes coding for resistance to the antibiotics ampicillin (amp) and tetracycline (tet) restriction sites p. BR 322 has restriction sites for a number of restriction enzymes including Bam H 1 p. BR 322 plasmids are incubated with the restriction enzyme Bam H 1 that ‘cuts’ the plasmid in the region of the tetracycline-resistance gene

Isolating and Locating Genes Both the human DNA and the p. BR 322 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 32 P (radioactive phosphorus) are used to synthesise the gene probe which in turn becomes a radioactive molecule

Making a Gene Probe Nucleotides containing 32 P are used to synthesise the gene probe Part of the ‘sense strand’ of DNA containing the nucleotide sequence that forms the gene for human growth hormone A chain of radioactive nucleotides, complementary to those of the gene sequence, is synthesised Radioactive gene probe that will hybridise (pair up) with the human growth hormone gene

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