Topic 8 B Flashcards
(16 cards)
What is the proteome of an organism?
All proteins that an organism is made up of
What is recombinant DNA technology?
Transferring a fragment of DNA from one organism to another, can be used to produce a protein in the cells of the recipient organism
How is reverse transcriptase used to make DNA fragments?
Cells that produce protein coded for by target gene will contain many mRNA molecules complementary to the gene so its easier to obtain. mRNA are used as a template strand to make DNA. mRNA is isolated from cells, mixed with nucleotides and reverse transcriptase. Reverse transcriptase uses mRNA to synthesise cDNA
How is restriction endonuclease enzyme used to make DNA fragments?
DNA contain palindromic sequences that are antiparallel. Restriction endonucleases recognise specific palindromic sequences. They cut the DNA at the recognition sequence which is complementary to the restriction endonucleases active site. Sticky ends are left which can be used to bind the DNA fragment to another piece of DNA has sticky ends with complimentary sequence using DNA ligase
How is a gene machine used to make DNA fragments?
Sequence is designed, first nucleotide in sequence is fixed to a support, nucleotides are added in the correct order adding protecting groups to make sure nucleotides are joined at the right points. Short sections of DNA (oligonucleotides- roughly 20 nucleotides long) are produced. They are broken from support and protecting groups are removed, The oligonucleotides are joined together to make longer DNA fragments
What is part 1 of using in vivo cloning to amplify DNA fragments?
Vector isolated, cut by restriction endonuclease (same used to cut DNA with target gene so same sticky ends are produced) Vector DNA and DNA fragment are mixed together with ligase (ligation), the new combination of bases is called recombinant DNA
What is in vivo cloning?
Gene copies made using living organisms
What is in vitro cloning?
Gene copies made outside of a living organism using polymerase chain reaction
What is part 2 of using in vivo cloning to amplify DNA fragments?
The vector with recombinant DNA is used to transfer gene into host cell. (transformed)
What is part 3 of using in vivo cloning to amplify DNA fragments?
Marker genes are inserted into vectors so host cell contains marker gene and gene to be cloned. Host cells grow on agar plate and replicates the DNA. Transformed cells will produce colonies with cloned gene and marker gene. Marker gene codes for antibiotic resistance and grow on agar plates with a specific antibiotic, Only transformed cells will grow or marker gene can be identified using UV light as it can code for florescence. Identified cells grow more producing lots of copied with the cloned gene.
How is in vitro cloning used to amplify DNA?
Mixture of DNA samples, primers (short pieces of DNA that are complementary to bases at start of fragment), free nucleotides and DNA polymerase is set up, mixture heated to 95 to allow the H bonds to break between two strands, then cooled to 50 so primers can attach. Heated to 75 so DNA polymerase can work and line up free nucleotides alongside and joins them. Two copies of fragment are formed and one cycle of PCR is complete. starts again until desired amount of DNA is obtained
How are alleles located using DNA probes?
DNA probes are complementary to base sequence of part of the target allele, probe has a label attached so it can be detected. DNA is digested into fragments using restriction enzymes and separated using electrophoresis. Fragments are transformed to nylon membrane and incubated with a fluorescent DNA probe, fluorescent band will show if gene is present
What is genetic counselling?
used to advise patients and relatives about risk of genetic disorders.
What can screening help to do?
Identify someone carrying a mutated allele, type they are carrying and the most effective treatment
How is genetic fingerprinting carried out?
PCR is used to make DNA fragments. DNA fragments are separated using electrophoresis, Gel is then placed under a UV light and DNA fragments are seen as bands
How does electrophoresis happen?
DNA mixture placed in well in a slab of gel and covered in a buffer solution, electric current passed through, DNA fragments negatively charged move to positive electrode at far end of gel, shorter fragments move further
