Making DNA Fragments
Reverse Transcriptase -Enzyme reverse transcriptase makes DNA from RNA molecule - produces complementary DNA (cDNA). mRNA isolated from cells, mixed with free DNA nucleotides + reverse transcriptase (uses mRNA as a template to synthesise a new strand of cDNA.
Restriction Endoneclease Enzymes - These are enzymes that recognise specific palindromic sequences (antiparallel base pairs) (recognition sequences) + cut (digest) the DNA in these places. Different restriction enzymes cut at different recognition sites (shape of recognition sequence, complementary to an enzyme's active site). DNA sample incubated with specific restriction enzyme, cuts fragment out via hydrolysis reaction. Leaves sticky ends- small tails of unpaired bases at each end of the fragment. Can be used to bind (anneal) DNA fragment to another fragment with complementary sequences.
Polymerase Chain Reaction (PCR) - Makes millions of copies of a fragment of DNA. A reaction mixture is set up that contains the DNA sample, free nucleotides, primers (short pieces of DNA that are complementary to the bases at the start of the fragment wanted) + DNA polymerase (enzyme that creates new DNA strands). Mixture heated to 95C to break hydrogen bonds, then cooled to 50-65C- primers can anneal to strands. Heated to 72C for DNA polymerase. Specific base pairing.
Gene Cloning (in vivo cloning - 1)
Definition: where the gene copies are made within a living organism. As the organism grows and divides, it replicates it DNA, creating multiple copies of the gene.
Process: DNA fragment inserted into vector DNA (vector= something used to transfer DNA into a cell. They can be plasmids or bacteriophages. Vector DNA cut open usin same restriction enzyme used to isolate the DNA fragment containing target gene. So sticky ends of vector are complementary to sticky ends of DNA fragment containing gene. Vector DNA and DNA fragment mixed together with DNA ligase (joins sticky ends of DNA fragmentto sticky ends of vector DNA). Process called ligation. New combination of bases in DNA is called recombinant DNA.
Gene Cloning (in vivo cloning -2)
- Vector with the recombinat DNA is used to transfer the gene into host cells.
- If a plasmid vector is used, host cells have to be persuaded to take it up. The plasmids are added and the mixture is heat-shocked (heated to 42C for 1-2 mins) - encourages cells to take in plasmids.
- With a bacteriophage vector, the bacteriophage will infect the host bacteriumby injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA.
- Host cells that take up the vectors containing the gene of interest are said to be transformed.
Not all cell take up vector + DNA. Marker genes can be used to identify transformed gene. Marker genes can be inserted into vectors at the same time as the gene to be clond. This means any transformed host cells will contain the gene to be cloned and the marker gene. Host cells are grown on agar plates and each cell divides and replicates its DNA, creating a colony of cloned cells. These all contain the cloned gene and the marker gene. The marker gene can code for antibiotic resistance - host cells are grown on agar plates containing specific antibiotic, so only transformed cells that have the marker gene will survive and grow. The marker gene can code for fluorescence - when the agar plate is placed under UV light only transformed cells will fluoresce. Identified transformed cells are allowed to grow more.
- Also known as recombinant DNA technology
- Organisms that have had DNA altered by genetic engineering are called trasformed organisms
- These organisms have recombinant DNA (DNA formed by joining together DNA from different sources
- Microorganisms, plants and animals can all be genetically engineered to benefit humans
- Transformed microorganisms can be made using the same technology as in vivo cloning
- E.g. foreign DNA can be inserted into microorganisms to produce lots of useful protein, e.g. insulin
- Transformed plants can also be produced - a gene that codes for a desirable characteristic is inserted into the plasmid. Plasmid added to a bacterium and the bacterium is used as a vector to get the gene into the plant cell. The transformed plant will have the desirable characteristic coded for by that gene.
- Transformed animals can be produced too - a gene that codes for a desirable characteristic is inserted into an animal embryo. The transformed animal will have the desirable characteristic coded for by that gene.
Benefits and Concerns of Genetic Engineering
Agriculture- Crops can be transformed so they give higher yields or are more nutritious - so reduce risk of famine and malnutrition. Can be transformed to have crop resistance - fewer pesticides needed - reduces cost + environmental damage. But farmers might create monoculture - makes crop vulnerable to disease because crop genetically identical. Also superweeds (resistant to herbicides) - would occur if transformed crop interbreed with wild plants.
Industry- Enzymes can be produced from transformed organisms - so produced in large quantities, less money = reduced costs. But without proper labelling people think they won't have a choice about consuming genetically engineered crop.
Medicine- Many drugs and vaccines produced by transformed organisms, using recombinent DNA technology - can be made quickly, cheaply + in large quantities. But companies who own genetic engineering technologies may limit use of technologies taht could save lives Also some people think use of technology could be used unethically (e.g. designer babies).
Genetic Fingerprinting (electrophoresis)
- Sample of DNA is obtained (e.g. from blood/saliva)
- PCR used to make many copies of the areas of DNA that contain the repeated sequence - primers are used that bind to either sides of these repeats and so whole repeat is amplified.
- End up with DNA fragments where the length corresponds to the number of repeats the person has at each specific position
- A fluorescent tag is added to all DNA fragments so they can be viewed under UV light
- The DNA fragment undergoes electrophoresis:
- The DNA mixture is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity
- An electrical current is passed through the gel - DNA fragments are negatively charged, so they move towards the positive electrode at the far end of the gel
- 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 - this is the genetic fingerprint.
- Two genetic fingerprints can be compared - e.g. if both fingerprints have a band at the same location on the gel it means they have the same number of nucleotides and so the same number of nucleotides and so the same number of sequence repeats at that placec - match.
Locating and Sequencing Genes
- DNA probes can be used to locate genes (e.g. on chromosomes) or see if a person's DNA contains a mutated gene. DNA probes are short strands of DNA. They have a specific base sequence that is complementary to the base sequence of the target gene. This means a DNA probe will bind to the target gene if it's present in a sample of DNA. A DNA probe also has a label attached, so it can be detected.
- As well as locating a gene, you can determine it's base sequence by restriction mapping. Different restriction enzymes are used to cut labelled DNA into fragments. DNA fragment are separated by electrophoresis. The size of of the fragments produced is used to determine the relative locations of cut sites. A restriction map of the original DNA is made - a diagram of the piece of DNA showing the different cut sites, and so where the recognition sites of the restriction enzymes are found.
- Gene sequencing is used to determine the order of bases in a section of DNA. A mixture is added to four separate tubes. It includes: a single-stranded DNA template, DNA polymerase (enzyme that joins DNA nucleotides together), lots of DNA primer, free nucleotides and fluorescently-labelled modified nucleotide. Tubes undergo PCR. The strands are different lengths because each one terminates at a different point. Separated by electrophoresis and visualised unde UV light. Complementary base sequence can be read of gel.
DNA Probes in Medical Diagnosis
DNA probes can be used to screen for clinically important genes (e.g. mutated genes). There are two ways to do this :
- The probe can be labelled and used to look for a single gene in a sample of DNA
- Or the probe can be used as part of a DNA microarray, which can screen lots of genes at the same time:
- A DNA microarray is a glass slide with microscopic spots of different DNA probes attached to it in rows
- A sample of labelled human DNA is washed over the array
- If the labelled human DNA contains any sequences that match any of the probes, it will stick to the array
- The array is washed, to remove any labelled DNA that hasn't stuck to it
- The array is then visualised under UV light - any labelled DNA attached to a probe will show up (fluoresce)
- Any spot that fluoresces means that the person's DNA contains that specific gene, e.g. if the probe is for a mutated gene that causes a genetic disorder, this person has the gene and so has the disorder
How it works: It involves altering the dfective genes inside cells to treat genetic disorders and cancer. How you do this depends on whether the disorder is caused by a muatated dominat allele (then you 'silence' it by sticking more DNA in the allele) or two mutated recessive alleles (then you add a working dominat allele)
How to get the new allele (DNA) inside the cell:The allele is inserted into cells using vectors. Different vectors can be used, e.g. altered viruses, plasmids or liposomes
Two types of gene therapy:
- Somatic Therapy= involves altering the alleles in body cells, particulary the cells that are most affected by the disorder. Somatic therapy doensn't affect the individual's sex cell (sperm or eggs) though, so any offspring could still inherit the disease.
- Germ Line Therapy= involves altering the alleles in the sex cells. This means that every cell of any offspring produced from these cells will be affected by the gene therapy and they won't suffer from the disease. Germ line therapy in humans is currently illegal.
Advantages and Disadvantages to Gene Therapy
- It could prolong the lives of people with genetic disorders and cancer
- It could give people with genetic disorders and cancer a better quality of life
- Carries of genetic disorders might be able to conceive a baby without that disorder or risk of cancer (only in germ line therapy)
- It could decrease the number of people that suffer from genetic disorders and cancer (only in germ line therapy)
- The effects of the treatm,ent may be short lived (only in somatic therapy)
- The patient might have to undergo multiple treatments (only in somatic therapy)
- It might be difficult to get the allele into specific body cells
- The body could identify vectors as foreign bodies and start an immune respose against them
- An allele could be inserted into the wrong place in the DNA, possibly causing more problems, e.g. cancer
- An inserted allele could get overexpresssed, producing too much of the missing protein
- Disorders caused by multiple genes (e.g. cancer) would be difficult to treat