PACK 22: GENE TECHNOLOGY Flashcards
(13 cards)
how can you obtain gene fragments
restriction endonucleases, gene machine, and reverse transcriptase
how do restriction endonucleases work
restriction endonucleases are complementary to go a specific recognition site on the gene, they hydrolyse phosphodiester bonds at the recognition site and produce fragments of DNA called sticky ends
what are sticky ends
single stranded sections of DNA that from an overhang at the end of a double stranded molecule
what does the gene machine do
an amino acid sequence is determined and from this the mRNA codons for each amino acid are looked up. the complementary DNA triplets are worked out and the gene is produced
what does reverse transcriptase do
an enzyme that converts mRNA to cDNA
what happens during the PCR process
at 95 degrees the hydrogen bonds in between the strands of DNA break and the strands of DNA are seperated. the temperature is reduced to 55 degrees and primers attach to complementary base pairs at the end of the single strands of DNA. taq polymerase attaches and the temperature is raised to 72 as this is the optimum temperature for this enzyme. free DNA nucleotides attach to the single stranded templates by complementary base pairs. DNA polymerase makes new strands by joining the free nucleosides together with phosphodiester bonds.
what is gel electrophoresis
the separation of DNA fragments based on their length and density, it can also be used to seperate protein molecules etc
what happens during gel electrophoresis
DNA samples are first amplified by PCR and then cut into fragments by the use of restriction endonucleases. the fragments are placed into wells at the end of a thin slab of gel. an electric current is passed through the gel, the DNA is attracted to the positive electrode due to the overall negative charge of DNA (due to phosphate groups). the molecules have to move through the gel, and shorter lengths of DNA move faster than larger lengths. so, the shorter the length of the DNA molecule, the further down the gel it will move in a given time.
what are DNA probes
a single stranded short sequence of DNA nucleotides with a complementary base sequence to the gene being screened for. it will behind to a target gene allowing identification. the probe has a label attached to it, usually a radioactive label of a fluorescent label
what is a genetic fingerprint
DNA contains repetitive sequences of DNA bases called variable number tandem repeats (VNTR’s), every individual has a different unique VNTR pattern- this is their genetic fingerprint
what is the process of in-vino cloning
the DNA fragment should be perpetrated for insertion, the DNA is then inserted into a vector (e.g. a plasmid), the DNA is then transferred into a suitable host cell (e.g. a bacterial cell) using the vector. identification of the transformed cells that have successfully taken up the gene using marker genes (e.g. antibiotic resistance). then comes the cloning of the population of transformed cells this is growth
what is gene therapy
the potential treatment for genetic diseases, involving altering the genotype
what is the process of gene therapy
tbe
