13. Techniques in Biotechnology Flashcards
what is biotechnology?
the use of cellular processes to make products of use to humans, including genetic testing, gene manipulation, cell replacement therapies, and tissue engineering
what is a genome?
a complete set of genetic information of an organism - 21 000 genes in human chromosomes
where is deoxyribonucleic acid found?
in the cells of all organisms in the nucleus, mitochondria, and sometimes the cytosol
what is primer?
a segment of dna complementary to the target sequence, initating replication with the presence of dna polymerase
what is dna polymerase?
an enzyme that facilitates dna replication
what is taq 1
an enzyme from the Thermal Aquaticus Bacterium that is an effective heat resistant DNA polymerase, allows heat to be used without denaturing the polymerase
what are restriction enzymes
enzymes derived from bacteria cut at certain sequence (the restriction site), producing either a straight cut/blunt end or staggered cut/sticky end
what is ligase?
it forms bonds between complimentary dna strands
- similar to polymerase, forming/facilitating an open strand
- useful recombining a double strand or piecing together broken parts
- acts across a nucleotide to bond complimentary nucleotides together
what is a synthetic nucleotide?
nucleotides made of a hydroxyl group, so it cant attach to the next nucleotide (terminates dna synthesis, purposefully ‘incorrect’)
What is electrophoresis?
a process used to organise dna by length so it can be profiled
what are vectors?
agents used to transfer dna from one cell to another
- plasmid: circular pieces of dna in a bacteria
- virus/phage: a virus that effects the bacteria and gives it the dna
what is dna sequencing?
the determination of the precise order of the nucleotides in a sample of dna
it is used to show whether a person will develop an inherited disease and changed allels can be detected
what is dna synthesised from?
four nucleotides (deoxynucleotide triphosphates)
how is dna synthesised?
- four separate sequencing reactions are set up in test tubes containing:
- multiple copies of single strand of dna to be sequenced
- dna primers
- normal free nucleotides
- small amount of dideoxynucleotides
- dna polymerase - dna polymerase will start replicating supplied dna sample by joining complementary nucelotides
- each time synthetic nucleotides attach to the growing dna strand, it will stop the elongation process- premature termination of synthesis
- eventually each reaction will result in a series of different sized dna fragments extending from the primers
- the final 4 mixtures are placed onto gel, and the different dna lengths move through the gel through electrophoresis alongside eachother
- fragments separate based on size, small and light fragments travel faster and further
- knowing what the final base must be for each fragment, the dna sequence can be determined by comparing the samples and base pairing
simple explanation of how dna is synthesised?
- the double stranded dna is converted to a single strand
- single stranded copies are placed in each four test tubes
- free nucleotides are added (phosphate, sugar, base)
- radioactive primers are added to each four test tubes, which bind to the single stranded dna and allow the complementary nucleotides to be added to the strands of dna
- altered nucleotides are also added and are labelled with fluorescent markers (genetic probe) -contains altered OH group, stopping dna synthesis
- Contents of the tubes are loaded into electrophoresis gel for separation, each into separate walls
- dna fragments separated by size, and electrophoresis bands can be used to determine the sequence of nucleotides on the original dna sequence
What is a hereditary spastic paraplegia?
inherited disorder that causes progressive limb weakness and stiffness, often resulting in paralysis
No cure, treatment involves physical therapy
Researchers searched for genes associated with the disease, located mutations on chromosome 2
Process of polymerase chain reaction
- denature:
- mixture of dna, spare nucleotides, primer, and dna polymerase is heated to ~95C
- separates the dna strand at the hydrogen bond (heat sensitive chemical bond) - Anneal:
- temperature is decreased to 55C
- Primers are added and join to the 3” end of the DNA strad - Extend:
- temperature increased to 72C
- taq dna polymerase adds bases complementary to the dna strand
what does PCR do?
shortens the time taken to detect hereditary diseases in a particular genome - each gene of interest can easily be amplified by pcr and then sequenced to detect the mutation in question
what occurs in electrophoresis?
- dna pieces are placed on a semi solid (agar) gel and an electric current is passed through electrodes at either end
- negative dna moves through pores in the gel to the positive electrode, smaller dna pieces move faster, and form a pattern of bands
what is a transgenic organism
those whose genome has been altered by the transfer of genes from another organism
what is a bacteriophage/phage
viruses that infect bacterial cells
what is more useful a blunt end or sticky end?
sticky ends as an overhang of a xfragment can be paired with any other dna fragment that has a corrseponding sequence, including a different organism
what are the benefits of a sticky end?
- ensure the dna fragment is inserted into the plasmid in the right direction
- requires less dna
- multiple sticky end restrcition enzymes can produce the same sticky end, even though each enzyme recognises a different sequence, increasing the likelihood that the target dna sequence can be cut by the enzymes
what is dna ligase
an enzyme form the E.Coli bacterium the ‘glues together’ short dna strands during replication
what is the method for producing an organism with recombinant dna
- isolate gene of interest, cut using restriction enzyme
- gene inserted into a vector and clones (vector has a segment of dna capable of replicating on its own)
- gene of interest is integrated into the vector and is referred to as recombinant dna
- cloned so that numerous copies are available to insert into host cells - after large quantities of the vector hva been produced, they can be integrated into host cells (usually bacterial, yeast, or mammalian cells)
- host cells will then produce the foreign protein using the instructions in the gene of the recobinant dna
how recombinant dna technology is used in producing insulin
introduced into bacterial cells, which are now cultured in vats where they mass produce insulin
- produce frequently performed using yeast cells as the growth medium
- doesnt result in any side effects or contamination risks as when patients were given inslin derived from the pancreas of pigs/cattle
how recombinant dna is used in producing human growth hormone
used to try and enhance athletic performance and to delay ageing
- has resulted in the production of growth hormone for dairy cattle
- administration of the hormone has increased milk production, research indicates that this milk doesnt pose a risk to human health- though animal ethical considerations still apply
how recombinant dna is used in factor VIII
haemophilia A is an inherited disorder that stops blood clotting, patients at risk of life-threatening bleeding
- injections of the protein concentrates made from human plasma have been used in the past requiring plasma from thousands of donors
- recombinant dna tech overcame this problem, and has the added benefit of being free from other proteins that could cause an immune response / allergic reaction
recombinant factor VIII is cultured in mammalian cells
how recombinant dna is used in vaccines?
vaccines are also produced using recombinant dna tech
- also has disadvantages- very expensive
- safety of product is paramount, so if a conventional vaccine is safe then there is little incentive to develop new ones
what is gene therapy?
aims to treat genetic abnormalities by replacing by faulty genes with healthy ones, correcting the underlying cause of a disease rather than simply treating the symptoms
What is cystic fibrosis?
- autosomal recessive
- results from mutations in the cystic fibrosis transmembrane regulator (CFTR) gene
- affects the lungs, pancreas, liver and reproductive organs
- characterised by thick sticky mucous secreted by mucous glands, which clogs the tiny air passages and traps bacteria in the lungs
- patients more susceptible to infection, which cause irreversible lung damage and shortens life expectancy
- prevents secretion of digestive enzymes from the pancreas (low fat, high carb diet advised)
How has gene therapy been used in cystic fibrosis?
scientists have successfully corrected faulty CFTR genes in cultured cells by adding normal copies of the gene to the culture
CF is a logical choice as it is a single gene disorder, the lungs are readily accessible for treatment, and the disease is slow to progress
first gene therapy treatment began in 1993 where researchers modified a common cold virus to act as a vector
What is huntingtons disease?
- single gene disease of chromosome 4, IT15
- mutated form of the huntington protein results in nerve cells becoming damaged
- characterised by mental/emotional changes, unintentional flailing movements of the arms and legs, and difficulty making voluntary movements of the limbs
- progressive dementia
what are stem cells?
undifferentiated cells capable of repeated mitotic division for long periods of time that have the capability of differentiating into specialised cells- candidates for producing replacement tissues
how are stem cells used in tissue engineering?
it restores healthy tissues or organs for patients, eliminating need for transplants or artificial implants
the use of stem cells overcomes issues of using cells of genetically diseased patients or from diseased organs
what are scaffolds in tissue engineering?
they serve as a template for tissue growth and have high pore sizes (enabling cells to grow while allowing diffusion of nutrients throughout the structure
What are scaffolds made of?
they are made of biodegradable structures so that they can be reabsorbed by surrounding tissues without being surgically removed
rate of degradation needs to match rate of tissue formation
what is the process of tissue engineering?
suitable stem cells are cultured
seeded onto the scaffold where they grow
cell covered scaffold is implanted into the patient - used for a wide range of tissues, eg. bone, skin, cartilage and adipose tissues
