Gene Technologies

Techniques to Study GenesPolymerase Chain Reaction (PCR)

Cutting out DNA fragments using Restriction Enzymes

Electrophoresis

Finding specific sequences of DNA using DNA Probes

Polymerase Chain ReactionThe PCR amplifies small DNA fragments which can be useful in forensic investigations. A reaction mixture is set up with the DNA sample, free nucleotides, primers* and DNA polymerase and heated to 95degC to break the hydrogen bonds between the two strands of DNA. The samples are now single stranded so the mixture is cooled to 55degC so the primers can anneal to the strands. ‘Taq polymerase’* is added to synthesise two new strands of DNA so it is now double stranded… the temperature is raised to 72degC so DNA polymerase can bind to this double strand (complementary base pairing). Two new copies are formed. This cyclic reaction can be repeated many times.

Using Restriction Enzymes (RE)

Some sections of DNA have palindromic* sequences of nucleotides; RE recognise specific palindromic sequences and cut the DNA at these places. Different RE cut at different specific points because the shape of the recognition sequence is complementary in shape to the enzyme’s active site. The DNA sample is incubated with the specific RE, which cuts the DNA fragment out via a hydrolysis reaction which breaks the sugar-phosphate backbone. Sometimes, this leaves sticky ends* which can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

ElectrophoresisA flourescent tag is added to all the DNA fragments so they can be viewed under UV light. The DNA is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity. An electric current is passed through the gel – DNA fragments are negatively charged because of its phosphoryl groups, so they move towards the positive electrode (anode). Small DNA fragments move faster and travel further through the gel, so the DNA fragments separate according to size. The DNA fragments are viewed as bands under UV light,

DNA ProbesA DNA probe is a short single-stranded section of DNA that is complementary to the section of DNA being investigated. A DNA probe will hybridise (bind) to the target sequence if it’s present in a sample of DNA. It has a label attached so it can be detected; the most common types are a radioactive marker (detected using X-ray) or a flourescent marker (detected using UV light). One use of DNA probes are to see if a family member has a mutation in a gene that causes a genetic disorder.

Terms*Primer a short piece of DNA that is complementary to the bases at the start of the fragment you want

‘Taq Polymerase’ an enzyme which is thermophillic so isn’t denatured by the hot temperatures used during the PCR

Palindromic antiparallel base pairs (read the same in opposite directions)

Sticky Ends small tails of unpaired bases a each end of the fragment

Sequencing the GenomeGenomes are firstly mapped. Samples of the genome are mechanically sheared into smaller sections and placed in separate Bacterial Artificial Chromosomes (BACs) and transferred to E-Coli cells. As the cells grow in culture, many copies are produced called clone libraries. Cells containing specific BACs are taken and cultured. The DNA is extracted and RE cut it into small, separate fragments which are separated during electrophoresis.

This allows genome-wide comparisons…

How?The identification of genes for proteins found in all or many living organisms gives clues to the relative importance of such genes to life

Comparing the DNA of different species shows evolutionary relationships

Modelling the effects of changing DNA can be carried out

Comparing genomes from pathogenic and similar non-pathogenic organisms identifies the genes that causes the disease so more effective drugs can be developed

Genetically Engineering a Microorganism

1. The DNA fragment containing the desired gene is obtained

2. The DNA fragment (with the gene in) is inserted into a Vector

3. The Vector transfers the gene into the bacteria

4. Identify the transformed bacteria

Steps 1 & 2…The DNA fragment containing the gene you want is isolated using RE. the DNA fragment is then inserted into a vector using RE and ligase. A vector transfers DNA into a cell; they can be plasmids or bacteriophages. The vector is cut open the same RE that was used to isolate the DNA fragment containing the desired gene so the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene. The vector DNA and DNA fragment are mixed together with DNA ligase which joins up the sugar-phosphate backbone of the two bits (ligation). The new combination of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA.

Step 3: Transferring the GeneThe vector with the recombinant DNA is used to transfer the gene into the bacterial cells. If a plasmid vector is used, the bacterial cells have to be persuaded to take in the plasmid vector and its DNA (e.g. they’re placed in ice-cold calcium chloride to make their cell walls more permeable). The plasmids are added and the mixture is heat shocked which encourages the cells to take in plasmids. With a bacteriophage vector, the bacteriophage will infect the bacterium by injecting its DNA into it. The phage DNA then integrates into the bacterial DNA. Cells that take up the desired gene are transformed.

Step 4: Identifying Transformed Bacteria

Not all bacteria take up the vector, so those that have can be identified using genetic markers. The genetic markers are inserted into vectors at the same time as the desired gene. The bacteria are grown on agar plates and each cell divides and replicates its DNA, creating a colony of cells. Transformed cells will produce colonies where all the cells contain the desired gene and the marker gene. The marker gene can code for antibiotic resistance, so the bacteria will be resistant to Ampicillin and Tetracycline. The agar plates contain the antibiotic so only cells with the marker gene will survive and grow.

Producing Human InsulinThe gene for human insulin is identified and isolated using RE. Specialised centrifugation methods separate mRNA from pancreatic tissue and reverse transcriptase uses this mRNA as a template to make single-stranded cDNA (made double by DNA polymerase). A single sequence of nucleotides are added to each end of the DNA to make sticky ends. A plasmid is cut open using the same RE. Plasmids and the insulin gene are mixed so sticky ends form base pairs. DNA ligase links the sugar-phosphate backbone of the plasmid and insulin gene making it recombinant. Plasmids are mixed with bacteria in the presence of calcium ions so some take up the plasmids and form a clone. Genetically engineered bacteria transcribe and translate the human gene to make insulin.

AdvantagesIt is advantageous to genetically engineer human insulin rather instead of using animal insulin…

• It’s identical to human insulin so it will be more effective and there will be less risk of a reaction

• It is a cheaper and faster way of providing a more reliable, larger supply of insulin

• No ethical or religious issues

‘Golden Rice’The psy gene (from maize) and the crtl gene (from the soil bacterium) are isolated using RE. A plasmid is removed from the Agrobacterium tumefaciens bacterium and cut open with the same RE. The psy and crtl and a marker gene are inserted into the plasmid; this recombinant plasmid is put back into the bacterium. Rice plant cells are incubated with the transformed A.tumefaciens bacteria, which infect the rice plant cells. A.tumefaciens inserts the genes into the plant cells’ DNA creating transformed rice plant cells. The rice plant cells are then grown on a selective medium so only transformed rice plants with the marker gene will be able to grow.

Advantages of Golden RiceThe resulting plants produce seeds with beta-carotene in the endosperm. Our bodies use beta-carotene to produce Vitamin A so ‘Golden Rice’ is being developed in countries such as south Asia and parts of Africa to help reduce Vitamin A deficiency. Vitamin A is needed for: bone growth; to form part of rhodopsin, a pigment needed for eyesight; maintenance and differentiation of epithelial cells which help reduce infection; synthesis of glycoproteins for cell growth and development. But, ‘Golden Rice’ has raised ethical issues and has said to reduce biodiversity by encouraging farmers to carry out monoculture which would leave the whole crop vulnerable to disease as they are genetically identical.

Gene TherapySomatic Cell Gene Therapy (SCGT):

• Augmentation (adding genes). Some conditions are caused by the inheritance of faulty alleles leading to the loss of a functional gene product. Engineering a functioning copy of the gene into relevant specialised cells means the polypeptide is synthesised and the cells can function normally

• Killing Specific Cells genetic techniques make such cells (e.g. cancers) express genes to produce proteins that make cells vulnerable to attack by the immune system

Gene TherapyGermline Cell Gene Therapy (GCGT) involved engineering a gene into the sperm, egg or zygote or into all cells of an early embryo so as the organism grows, every cell contains a copy of the engineered gene. Here, the genetic modification is restricted to somatic (body) cells with no effect on the germline. An individual can still pass on the allele for the disorder to their offspring. This gene therapy, although legal, is ethically unacceptable for fear it could create a new disease or interfere with evolution.

Somatic Germline

The functioning allele of the gene is introduced into the target cells

The functioning allele is introduced into germline cells so delivery techniques are more straight forward

Treatment is short lived and has to be repeated. The specialised cell will not divide and pass onto the offspring

All cells derived from the germline will contain a copy of the functioning allele. It can be passed onto the offspring

Difficulties in getting the allele into the genome in a functioning state

It is unethical to engineer human embryos

Issues with Genetic Engineering

Some people are worried that using antibiotic-resistance genes as marker genes may increase the number of antibiotic-resistant, pathogenic microorganisms

Genetically engineering animals for xenotransplantation (transfer of cells/tissues/organs from one species to another) may cause them suffering

Some are concerned about ‘superweeds’ – weeds that are resistant to herbicides as they’ve bred with genetically engineered herbicide-resistant crops

What is humans are genetically engineered and a genetic underclass is created? (This is currently illegal)