Year 13 - Gene tech Flashcards
(14 cards)
What is the advantage of using a gene machine over reverse transcriptase to isolate a gene
Faster to use gene machine than all the enzyme catalysed reactions with reverse transcriptase
what is the advantage of using gene machine/reverse transcriptase over using restriction endonucleases to isolate a gene to put into a bacteria cell
Endonucleases isolate gene from human DNA
Human gene contains introns
Bacteria cannot remove introns by splicing.
Reverse transcriptase/ gene machine produces gene without introns
What is the advantage of using reverse transcriptase to isolate a gene for insertion into a vector?
mRNA is much easier to obtain as finding cell producing large amounts of proteins will also have many mRNA molecules.
Mature mRNA can be reverse transcribed to DNA fragments that doesn’t contain introns so will code for functioning protein without need for splicing
Describe the process of inserting a gene into a plasmid/DNA
- cut plasmid and gene with same restriction endonuclease to produce ‘sticky ends’
- both have complementary stick ends that can anneal by hydrogen bonding between complementary base pairs
- mix with DNA ligase to join sticky end parts of gene and plasmid with phosphodiester bonds
why do scientists aim to insert a vector (plasmid with required gene) into cytoplasm or circular DNA of bacteria but nuclear DNA of humans
Bacteria divide by binary fission that copies plasmids and circular chromosome that contain desired gene to produce genetically identical cells
But
Human cells divide by mitosis which will only copy desired gene if it is in nuclear DNA to produce genetically identical cells. Plasmids are not copied and passed on in mitosis
When inserting a vector (plasmid containing gene) into eukaryotes, why do scientists inject into gametes/zygotes and not somatic (body) cells
Zygote cell is totipotent so divide by mitosis and differentiate into all cells of organism.
Therefore gene will be in cell that will transcribe gene into mRNA and translate mRNA into named protein
Explain the purpose of attaching a gene such as GFP jelly fish gene alongside the gene of interest into the vector that is inserted into embryo/zygote
Not all cells will successfully take up the plasmid
GFP gene is a marker gene to show transformed cells and what cells are expressing the desired gene.
As only cells/embryos will show fluorescence and glow under UV light
Describe in vivo gene cloning
- Cut desired gene from DNA of organism with a restriction endonuclease
- Cut plasmid and DNA fragment sample with same restriction endonuclease.
- This produces the same sticky ends on DNA fragment and plasmid
- DNA ligase joins DNA fragment and plasmid to form recombinant DNA
(give a heat or electrical shock to open up small pores in cell surface membrane of named cell) - Recombinant plasmid taken up by bacteria.
- Bacteria replicate DNA by semiconservative replication and divide by binary fission
Describe the polymerase chain reaction (5 marker)
Heat DNA (94-98 degrees) to separate strands by breaking hydrogen bonds.
Add primers (short ssDNA sequences) that has specific base sequence that is complementary to start of allele (whatever dna is to be copied).
Add free DNA nucleotides.
Cool temperature (50-64 degrees) to allow binding of primer and nucleotides to DNA by complementary base pairing with H bonds.
Increase temperature (72-80 degrees) to allow heat stable taq DNA polymerase to join adjacent DNA nucleotides with phosphodiester bonds in condensation reactions to produce complementary DNA
Repeat cycles many times.
Define DNA probe and explain why you may make them dyed/ radioactive
Short single strand of DNA that has a base sequence complementary to known base sequence of DNA (such as a gene) DNA is invisible so radioactive nucleotide/dye gives out light to allow for detection of the location of gene on the chromosome the probe is attached to
When looking for a probe cells in mitosis were used, why?
Cells in mitosis have visible chromosomes as they condense during prophase.
Can see which chromosome DNA probe is attached to.
How do you screen DNA for multiple DNA sequences of interest/genes
amplification: Use PCR to replicate many copies of DNA
Cut DNA using restriction endonucleases
Separate DNA fragments using gel electrophoresis
Add a labelled DNA probe that binds by DNA hybridisation
Shine a UV light to identify DNA of interest//genes by fluorescence
Describe how genetic fingerprinting can be carried out on a sample of DNA and why it can be used to identify a person
Extract DNA from sample and cut using restriction endonuclease to make blunt end fragments.
Cut must leave VNTRs (variable number tandem repeats) intact
Separate DNA fragments according to length using electrophoresis
By placing sample into well on gel and passing electric current through.
Use alkaline solution to make DNA single stranded
Use southern blotting to transfer to nylon membrane.
DNA hybridisation with added radioactive DNA probe and target fragment.
Use x-ray film to see
Pattern unique to individual
Name and explain factors may slow down the process of obtaining a genetic fingerprint
if only Small amount of DNA obtained -> needs PCR for amplification -> PCR increases amount of DNA so enough available for genetic fingerprinting.
If DNA sample is contaminated with other DNA present -> need to identify required DNA from rest and then amplify using PCR
