Biotechnological techniques HBS3B

Biotechnological techniques Biotechnological techniques are being developed and used for identification of hereditary diseases by DNA sequencing profiling techniques PCR (polymerase chain reaction) genetic probes production of human proteins, hormones and vaccines by DNA recombinant techniques (including restriction and ligase enzymes) e.g. to produce insulin, Human Growth Hormone, Factor VIII treatment of genetic disorders by gene therapy e.g. cystic fibrosis cell replacement therapy and tissue engineering by the cloning of stem cells e.g. repair of injured tissues, treating degenerative nerve diseases.

Human Genome project This is a project that set out to identify the complete sequence of nucleotides in human DNA Although the sequencing has been completed, the analysis still continues The project attempts to map locations of known genes on the chromosomes. At present the locations of about 4000 genes responsible for inherited disease have been identified Possible uses for the data collected are identification of presence of genetic diseases in individuals (diagnostic testing development of gene replacement therapies aid research into disease progression (eg identifying promoter or regulator genes)

DNA sequencing DNA sequencing involves identifying the precise order of nucleotide in DNA. In DNA profiling sequences are compared between individuals (DNA fingerprint or profile) Special enzymes called restriction enzymes are use to split the DNA into segments so the sequence of nucleotides can be identified. It is used to identify the genetic relationships between individuals (eg to confirm paternity) forensic science (eg identifying criminals) look for genetic disease

Gel electrophoresis Gel electrophoresis refers to a process that allows identification or observation of DNA patterns Uses include identification of individuals from samples (eg forensic science) genome sequences presence of inherited disease

Sequencing by electrophoresis

Polymerase chain reaction Polymerase chain reaction (PCR) refers to the process used to replicate DNA. This increases the amount of DNA present in a sample. It is needed so that there is enough DNA present to test.

Genetic probes Probes are nucleic acid bases arranged in a sequence which is complementary to a sequence in the genome They usually have radioactive or chemical markers that are used to make them easily visible They can be used to detect if a genomic sequence is present, deleted or altered. They are often used to identify the presence of a gene for a disease

Recombinant DNA technology This is also known as genetic engineering This refers to the processes that allow introduction of new DNA into a cell. This allows the taking of genes from one organism and placing them in the chromosomes of another producing a transgenic organism. The genes can come from individuals of the same species (eg transferring a healthy gene from one person into a person with a genetic disease = gene therapy) or from a different species (eg placing insulin producing gene into a bacteria) Uses include gene therapy, producing transgenic bacteria that can produce useful products (eg insulin). Products now being produced by this technology include hormones (eg insulin, hGH, FSH), factor VIII or vaccines Some risks and concerns associated with recombinant technology include cost, religious objections (‘tinkering with God’s work’), risk of cross species diseases or diseases resistant to treatment, unknown side effects

Terms used in recombinant DNA 1 Vector - Bacterial plasmids, viral phages or other such agent used to transfer genetic material from one cell to another Phage or bacteriophage - a virus that infects bacteria Plasmids - Small circular strands of DNA distinct from the main bacterial genome. They are composed of only a few genes and are able to replicate independently within cells

Terms used in recombinant DNA 2 Restriction enzyme - Enzymes that cut strands of DNA at specific sequences of nucleotides Ligase - An enzyme that is capable of combining two small components of single-strand DNA into one single structure. Straight cut - Produced when a restriction enzyme makes a clean break across the two strands of DNA so that the ends terminate in a base pair; called blunt ends Blunt ends - The ends produced by a straight cut of a sequence of nucleotide bases Staggered cut - Produced when a restriction enzyme creates fragments of DNA with unpaired nucleotides that overhang at the break in the strands; called sticky ends Sticky ends - The overhanging ends produced by a staggered cut of a sequence of nucleotide bases; sometimes called cohesive ends

Transfer and cloning of insulin gene

Recombinant DNA techniques Restriction enzymes are used to cut sections of DNA at specific locations and are necessary because they allow the selection of 1 gene or DNA fragment Ligation refers to joining of the two segments and is necessary because it produces 1 strand of DNA or a plasmid now containing the gene A gene for antibiotic resistance is usually added so that the bacteria with the new DNA can be identified and collected

Gene therapy Gene therapy involves insertion of DNA to replace faulty genes It is used when a single defective gene can be identified and a healthy one is available eg cystic fibrosis, Huntington’s disease, muscular dystrophy, diabetes Germ line therapy refers to replacement of DNA in sperm or ova and therefore aims for treatment that will be passed on to offspring. It has not been attempted due to the difficulty and the ethical considerations of changing the human genome Somatic cell gene therapy refers to replacement of DNA in non-reproductive body cells and therefore aims for treatment that will be effective in the sufferer, but can not be passed on to offspring.

Gene therapy In ex vivo gene transfer, genes are transferred into cells grown in culture. The cells are then transferred back into the patient. This is done when tissues can be removed and grown outside the body without harm to the patient or the tissues (eg muscle, skin) In in vivo gene transfer, genes are transferred directly into the tissues of the body in the patient. This is done when tissues can not be removed and grown outside the body without harm to the patient or the tissues (eg brain)

Inserting genes Introduction of new genes can be done by viral or non-viral means. With viral means, DNA is inserted into a virus which is then injected into the tissue culture or patient. When the virus infects the cells it alters their DNA – thus adding the replacement gene

Inserting genes 2 One problem with viral insertion is that the immune system may destroy the virus before the gene has been transferred successfully. Another problem is the risk of disease if all disease causing genes have not been removed from the virus Non-viral means include direct injection, where DNA is injected directly into cells One problem with this is it requires large amounts of DNA and can only be used on certain tissues the use of liposomes where lipid spheres with watery core containing the DNA are injected. These fuse with cell membranes and the DNA passes through the membrane into the target cells by introducing an artificial chromosome (chromosome 47) which contains a number of replacement genes, and would be copied every time the cell divided One problem with this is the difficulty of getting such a large molecule into the cell. There are some issues associated with gene therapy. These include cost, technical difficulty, religious objections (‘tinkering with God’s work’), risk of new diseases or diseases resistant to treatment, unknown side effects

Delivery techniques