Gene Technology

Gene Technology Jargon

Gene technology: a general term covering a variety of technics involving genetic material including genetic engineering, creation of gene libraries & DNA frequencing.

Genetic engineering: the transfer of a gene from one organism (the donor) to another organism (the recipient) e.g. the gene of insulin

Gene Technology Jargon

Restriction enzyme/ restriction endonuclease: an enzyme which cut the DNA at a particular site determined by the base sequence.

Marker: an easily recognised gene which is transferred along with the target gene during genetic engineering e.g. fluorescent pigments

Gene Technology Jargon

Promotor: a section of DNA directly before the gene where RNA polymerase attaches to the DNA allowing transcription to begin.

Vector: a carrier DNA molecule which allows the gene which is to be transferred to be inserted into the recipient organism e.g. viruses or bacterial plasmids

Gene Technology Jargon

Genetic fingerprinting: an analytical process which is used to determine the relativeness of two DNA sample.

DNA sequencing: the process which determines the precise sequence of nucleotides in a sample of DNA

Synthesise human insulin

Why produce human insulin– It is identical to human insulin therefore liver cell

s react more quickly, the hormone is more easily broken down, and does not elicit an immune response.

– Some religions groups e.g. Muslins & Jews might object to use pig insulin

– It avoids contamination e.g. prion proteins Bovine spongiform encephalopathy

Synthesise human insulin

Using internet find out the stage of synthesis human insulin. – 15 mins

Using the information which you find, write down the stages. – 15 mins

Present you work – 30 mins Remarks: detail stages, including which RE it

use, which vector it use. etc.

Stage 1: Identification and isolation of the gene. Pancreas cells were used because they contain la

rge amounts of mRNA. mRNA for the insulin gene was isolated and purified. This mRNA has the advantage that the junk DNA sequences (introns) have already been removed.

The mRNA was incubated with DNA nucleotides and a reverse transcriptase to make DNA i.e. the insulin gene

Then the single strands of DNA are incubated with DNA polymerase to produce double strands.

Stage 2: Short sequences of single strand DNA are

added to the ends of the gene. These sticky ends correspond to other sticky ends produced when DNA is cut by a particular restriction enzyme. e.g. ecoR1

The restriction enzyme which is chosen must not cut the gene i.e. the sequence must not appear in the DNA sequence of the gene.

Sticky end

Stage 3: DNA plasmids from bacteria are cut using

the same restriction enzyme. Then the genes are incubated along with the plasmids and hopefully the gene DNA will become inserted into the plasmid DNA.

NB: a plasmid is the natural method by which bacteria transfer genes between one another. As such, it allows us to transfer genes into the bacterial genome, because the bacteria have a natural tendency to acquire plasmids.

Stage 4:

Plasmids containing the human genes are mixed with bacteria e.g. Escherichia coli (E.coli) and incubated. Some of the bacteria will take up the plasmid and incorporate it into there own genome.

Stage 5: The bacteria are spread over agar plates

and allowed to grow. Then bacterial colonies which have incorporated the insulin gene are identified. Originally, this was done by transferring a sample from each colony onto another agar plate which contains antibiotics for which the resistance gene was transferred along with the insulin gene as a marker. Only the bacteria with the insulin gene will grow. These can be separated and cultured in large quantities.

Issues associated with genetic engineering

1. Promoters: are necessary to regulate the transcription of genes. The original genetic engineering involving insulin used the lac operon. The lac operon act as follows.

– Lac operon – a system of promoter & gene for breaking down lactose – promoter & gene for β–galactosidase enzyme & gene for lactose permease.

Issues associated with genetic engineering

There is a protein know as the lac repressor which attaches to the DNA next to the promoter sequence preventing the attachment of RNA polymerase. However in the presence of lactose, lactose molecules attach to the repressor changing its configuration so that it no longer attaches to the DNA. This allows RNA polymerase to attach & express the gene. So in the original experiment, the bacteria would produce insulin protein in the presence of lactose.

Issues associated with genetic engineering

2. Markers: originally antibiotic-resistant marker were used. However, there are 2 problems using these two markers.

The antibiotic-resistant genes could spread to pathogenic species. e.g. cholera

Antibiotic resistance occurs naturally in bacteria therefore it is possible for resistance strains to not contain the insulin gene.So nowadays, genes which are harmless and not naturally occurring in bacteria are used. e.g. fluorescent protein from jellyfish – glows under UV light or a gene for substances which are easily stained.

Issues associated with genetic engineering

3. Eukaryotic organisms: insulin is a simple protein which could be successfully produced by bacteria. However, large proteins with more complicated configurations can only be produced by eukaryotic organisms. So for these proteins, we would use single-celled eukaryotic organisms e.g. yeast

DNA fingerprinting This process allows us to analyse and identify i

ndividuals from samples of DNA. DNA finger printing involves cutting the DNA into fragments using restriction enzymes. Different people have different DNA especially highly repetitive nonsense sequences or VNTR (variable number tordem repeat). These sequences are different but also inherited e.g. someone may have 50 or 150. The variable number means the size of the fragments in different individuals is variable.

Stage 1

DNA is extracted from a sample of tissue. This DNA is treated with specific restriction enzymes. Chosen because around the repetitive sequences.

Stage 2 Electrophoresis DNA molecules

are negatively charged and therefore move towards the anode. They become separated into bands because the different size molecules move at different speeds.

Stage 3

When electrophoresis is complete, a layer of nylon is laid on top of the gel. The absorbent material e.g. tissue is pressed onto the nylon. The tissues absorbs water from the gel by capillary action in doing so the DNA fragments become attached to the nylon membrane.

Stage 4

The DNA is heated cause denaturation i.e. the two strands separate. Then a DNA probe is added. This is a short single strand of DNA containing radioactive phosphorus. It attaches to the exposed DNA fragments in locations where the DNA sequence matches.

Stage 5

A piece of X-ray film is placed over the nylon sheet and as the radioactive isotope decays, the radioactivity released, creates a pattern of lines on the film.

DNA sequencing This process involves determining the

sequence of bases found in a molecule of DNA.

DNA sequencing

The DNA molecule is multiplied, then broken down are base at a time. The remaining fragments vary in lengths from one base to the whole molecule can be separated by electrophoresis. A second process of electrophosesis can determine which base is on the end of each fragments and therefore the entire sequence can be deduced.

Most recently, DNA sequencing has been used in a large scale in the human genome project (HGP). This involve sequencing of the entire human genome so as to identify genes and mutations.

Gene therapy – e.g. cystic fibrosis Gene therapy is a process which aims to

cure or prevent disease by changing the genotype.

There are 2 potential approaches:1. Germ cell therapy i.e. sperms, eggs, zygotes.

This has the advantage that all the cells in the body derived from the germ cells will contain the same alteration. However, it is not carried out for ethical reasons since the potential beneficiary is unable to consent to the procedure.

Gene therapy – e.g. cystic fibrosis2. Somatic cell therapy. This involves

changing the genotype of body cells by adding/removing a particular gene. In the case of cystic fibrosis, this is made easier because it is recessive, and therefore only requires a functioning copy of the gene into the nucleus of the cell to allow the production of the ion channels. Somatic therapy has 3 main drawbacks:

Gene therapy – e.g. cystic fibrosis

3 main drawbacks– The gene must be inserted into all somatic cells

individually.– The difficulty of delivering the gene.– The limited lifespan of the affected cells.

For cystic fibrosis it is estimated that less than 10% of the epithelial cells needs to be functioning to reduce the symptoms dramatically.

Gene therapy – e.g. cystic fibrosis3. Methods of delivery have been attempted:

Virus – e.g. Adenovirus. Viruses are specialized to deliver DNA into cells. If the harmful genes of the virus are replaced by the chloride transport protein genes, the genes can be incorporated into the cells’ genome. The virus can be delivered through an aerosol.

Liposome – A sphere of lipid surrounding the DNA which when introduced through aerosol will fuse with cell membrane introducing the gene into the cells.

Gene therapy – e.g. cystic fibrosisMicroinjection – Physical injection of DNA into

the cell. This is only practical in laboratory situation.

Real trials of gene therapy were carried out in Jam, 1985 using a liposome delivery system. Viruses were not used because they stimulate an immune response. The study produced a 20% improvement in potential difference a cross the cells of the nose lining. However, the effect lasted for about a week only.

Genetic screening

This is the process by which samples of DNA by individuals are tested for the presence or absence of individual alleles and therefore the risk of passing on an inherited condition. Screening can take place at 3 stages:

Genetic screening – 3 stages1. Before pregnancy – obviously at this stage we will scree

n the parents looking for recessive alleles e.g. cystic fibrosis. A DNA probe labelled with radioactive markers can be added to the DNA and will attach to the mutant gene if it is present.

2. During pregnancy – looking at the embryo or the fetus's DNA. There are 2 techniques for gathering cells from embryo

Amniocentesis – a hypodermic syringe is inserted into the amnion carefully avoiding the fetus. A sample of amniotic fluid is withdrawn which contains cells from the fetus which can be cultured and tested.

Chorionic villus sampling – a catheter is inserted through the virgina to the uterus gathers cells from the placenta.

3. After pregnancy – e.g. PKU(phenylketonuria) are tested from a blood test on the new-born infant.

Genetic counseling The process of genetic screening can produce

worrying and upsetting results therefore genetic counseling is used to try to help people deal with the results of genetic screening. The kinds of questions involved are:– Is it right to have the test– What are the risks associated with becoming

pregnancy.– What action can be taken e.g. selection of embryo

from I.V.F or abortion.– The effects of the genotype of the offspring.

Discuss the possible benefits & dangers of gene technology

Practical Ethical Social