3.5.8 Gene Cloning technologies allow study and alteration of

gene function in order to better understand organism function

and to design new industrial and medical processes

Genetic engineering

• Can allow genes to be manipulated, altered and transferred from organism to organism

• Why might this be useful?

• One use has been to produce human chemicals such as insulin.

• When the DNA is introduced into a new organism and combined with its own it is known as recombinant DNA and the organism is known as a Genetically modified organism (GMO)

The process of making proteins using DNA technology:

• Isolation – getting some desired DNA• Insertion – putting the DNA into a vector• Transformation – inserting the vector into a

host• Identification – making sure that the host

has taken up the DNA• Growth/Cloning – getting a large population

of host cells

Isolation - How to get some DNA fragments!

• If the amino acid sequence of the desired protein is known, the DNA code can be worked out and the DNA made in the lab by stringing together the correct order of nucleotides.Note:Many proteins are extremely large, therefore this would be a tedious process.

• Conversion of mRNA to cDNA, using reverse transcriptase.

• Cutting DNA at specific palindromic recognition sequences using restriction endonucleases.

Conversion of mRNA to cDNA, using reverse transcriptase.

• Activity:– Now that you know

the correct sequence complete the cut and stick worksheet to put the synthesis of cDNA into the correct order

– Add any extra details about the process which is occurring e.g. splicing and the information on page 247

Interesting fact

• The technology for producing cDNA is actually used by HIV

Cutting DNA at specific palindromic recognition sequences using restriction endonucleases.

• A restriction enzyme (or restriction endonucluease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites, which are usually 4 – 6 base long

Cutting DNA at specific palindromic recognition sequences using restriction endonucleases – Continued

• Such enzymes, in bacteria and archea, are thought to have evolved to provide a defence mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity.

• To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

Blunt or sticky!

Blunt or sticky!

• Sometimes a straight cut occurs this is known as a blunt end.

• Sometimes a staggered cut occurs, which leaves and uneven cut in which the DNA strand has exposed unpaired bases – known as a sticky end.

• If you read the unpaired bases each from left to right they are opposites of one another, i.e they are a palindrome.

But how do you know where to find the desired gene in the first place?

• Using a genetic probe – You know the DNA base sequence of the gene for the desired protein so a section of base sequence can be radioactively labelled.

• This section of DNA with the correct base sequence is called a probe.

• The DNA is "unzipped" so that it becomes single stranded and a probe would anneal (attach) if there were complementary bases.

• The probe is added and sticks to the correct complementary fragment. The correct fragment can now be identified, as it is radioactive.

Now is time to create lots of copies of the isolated DNA

• There are two ways to get lots of clones of the DNA sequences which has been isolated:– In vivo – cloning by transfering the DNA

into a host cell using a vector and the host copying the DNA.

– In vitro – using polymerase chain reaction (PCR)

In vivo gene cloning using vector

What type of organism would make a good host?

1. Grows fast. 2. Is easily manipulated. 3. Has a simple chromosome (prokaryotic

cells do not have a nuclear envelope).

4. Contains naturally occurring vectors (see later).

• A good option therefore is to use yeasts or bacteria.

How to get the DNA into the Host – use a vector!

• A vector is a carrier DNA molecule into which the desired gene can be inserted.

• Most commonly, this vector is a plasmid. This is a small, extra-chromosomal, circular piece of DNA often found in bacteria in addition to their functional DNA.

The plasmid - • The plasmids are modified so that they have

two or more genes for resistance to antibiotics.

• They should also contain a sequence that can be recognised by the same restriction enzyme used to cut the fragments. This enables sticky ends to be complementary – why do you think this would be useful?

• The site that is cut should be in one of the genes for antibiotic resistance.

• A Plasmid:

Importance of Sticky Ends

• Using pages 249 – 250 explain the importance of sticky ends – use diagrams to help you.

Step 1 – Cut the plasmid and the Desired DNA

• Cut the genome with a restriction enzyme (RE) and mix with the plasmid that has also been cut with the same R.E so that the sticky ends of the fragments and the plasmid are complementary.

• Hopefully, some fragments will insert into the plasmid DNA before either segment joins with itself.

• The join is made permanent by DNA ligase

The fragments are added to the plasmids with these possible outcomes:

1. Plasmid rejoins, tetracycline gene now intact.

2. Fragment joins with plasmid. Tetracycline resistance gene is interrupted; the fragment does not contain the desired gene.

The fragments are added to the plasmids with these possible outcomes:

3. Fragment joins with plasmid. Tetracycline resistance gene is interrupted; the fragment does contain the desired gene.

4. The fragment joins with itself. .

Now it can be introduced into the host

• Transformation – re-introducing plasmids to bacterial cells

• Mix the bacterial cells together with the plasmids and some calcium ions.

• Calcium ions and changes in temperature make the cell membrane of the bacteria permeable and allow the plasmid to pass through.

• Only about 1% will have taken up the correct plasmid.

Identifying the transgenic bacteria – with the introduced gene in the correct place!

• The bacteria are transferred to a plate containing the antibiotic ampicillin.

• Those bacteria that have taken up any plasmid will be resistant to the antibiotic so will survive and form colonies.

• Those that have not taken up the plasmid will not be resistant and die

Is this enough to make sure that the gene is present?

What would you do next?

Use the genetic marker – Antibiotic resistance• These colonies are then

replicated onto plates containing the antibiotic tetracycline.

• Those bacteria with recombinant plasmids will not survive because the fragment has disrupted the gene for resistance.

• The 2 plates are compared and those colonies resistant to ampicillin but not to tetracycline can be identified. All these colonies contain recombinant plasmids.

Can you see any problems

with this process?

Pg - 252

Other markers

• A fluorescent protein – The gene can be inserted into the green fluorescent protein gene, this means that the bacterial which cannot glow in the dark have not taken up the plasmid.

• An enzymes marker – lactase enzyme, it can turn a colourless substrate blue – this means if grown on the substrate those that have not taken up the plasmid with the gene inserted into the lactase enzyme gene they will turn it blue. – Benefit – quicker because you do not need to

carry out replica plating because the colonies you need are not killed.

In vitro – using polymerase chain reaction (PCR)

• PCR – a rapid efficient method of copying fragments of DNA

• It required the following:– DNA fragments– DNA polymerase – it’s extracted from bacteria

which live in hot springs – useful because it will not denature at hot temperatures

– Primers – Nucleotides– Thermocyler – a computer controlled machine

that varies temperature precisely over a period of time

In vitro – using polymerase chain reaction (PCR)How the PCR works:• There are three steps, repeated for up to 40 cycles in an automatic

cycle, which heats and cools the reaction mixture very rapidly.1. Separation of DNA - The DNA strand is heated to 95°C, to

denature it and open the strands, forming single strands.. 2. Annealing of primers - at 55°C. During this process the primers

are jiggled around by molecular collisions (Brownian motion). Ionic bonds are formed and broken between the single strands of primer and the template. In the areas where more exact fits are made, the bonds last longer, allowing the DNA polymerase enzyme to start copying the template. (The heat stable TaqDNA polymerase comes from a thermophylic bacterium found in hot springs.) The primers also prevent the two orignal strands from joinging

Note: Very pure DNA building blocks dCTP,dATP,dGTP and dTTP (one for each of the four nucleotide bases) are in the machine at the start.

3. Sythesis of DNA - Extension at 72°C. This is the optimum temperature for the DNA polymerase. Here the bases complementary to the template are coupled to the primer on the 3’ side.

Note: Because both strands are copied during the PCR process, the rate of increase is exponential.

Click me!

Activities

• Using page 255• Task 1 – Draw out figure 2 to aid with

your understanding of PCR• Task 2 – Using the information on the

page draw up a table summarizing the advantages of in vivo and in vitro cloning of DNA

Using recombinant DNA technology

• Your task – – You have been asked to write an article for

Biological science review about how recombinant DNA technology can be used and the ethical, moral and social issues related to its use.

– Due date – 20/04/10 – Remember you miss you lessons next week so this has to reflect the time allowed.

– Length – at least 3 – 4 sides of types work with images.

– Bibliography of resources used must be included.– Content – must cover use in micro-organisms,

plants and animals. – Your book is a good start point but it is only a start

point!