Gene Technology Flashcards
Explain why in gene technology a promoter needs to be transferred along with the desired gene
- Promoter initiates transcription/ switches on gene/ causes gene expression
- Binding of RNA polymerase/ transcription factors
- otherwise gene has to be inserted near an existing promoter
- this is difficult to do/ may disrupt expression of existing gene
- in eukaryotes, precise position of promoter is important
Explain how DNA sequencing could be used to compare the DNA of modern-day and ‘regenerated’ S.stenophylla
- Total DNA genome cut into fragments
- By restriction enzymes
- DNA denatured/ made single stranded
- primers used/ polymerase chain reaction (two PCR primers are needed in a PCR reaction. Primer = short nuclei acid sequence that provides a starting point for DNA synthesis)
- chain termination (dideoxynucleotides? = chain-elongating inhibitors of DNA polymerase)
- DNA/ Taq polymerase (type of DNA polymerase enzyme)
- copies of different lengths produced
- electrophoresis
- detection of fluorescence by laser scanner
- sequence of bases/ nucleotides read by computer
Outline the principles of electrophoresis as used in sequencing this DNA
Cut DNA into fragments using restriction enzymes
Place on agarose gel
Apply current
Fragments travel towards anode (positively charged electrode bc DNA negatively charged)
Short fragments travel further than long ones as less impeded by gel
Visualise DNA with UV light
Southern blotting used
State 2 advantages of treating diabetes with insulin produced by gene technology
Identical to human insulin More rapid response Fewer rejection problems Ethical reasons Less risk of transmitting disease
Explain importance of plasmid having a single target site for a particular restriction enzyme
Single target site will be in correct resistance gene
Gene to be inserted has complementary sticky ends to target site sticky ends
More cuts would fragment plasmid
Explain why genes for antibiotic resistance are now rarely used as markers in gene technology
Risk spreading resistance to other bacteria
Spread of resistance makes the use of antibiotics less effective
Resistance spread via conjugation/ transformation/ uptake of plasmids
Plasmid multiple resistance
Describe the use of alternative marker gene that can be used instead of an antibiotic gene
Gene for fluorescent substance
Source of gene = jellyfish
Substance fluoresces when exposed to UV light
LacZ gene/ gene for B-galactosidase
Splits non-blue substrate
Product is blue
Explain why plasmids are well suited for use as genetic vectors
Small so can be inserted into cells
Replicate independently so large number of plasmids (have origin of replication)
Has restriction site so new gene can be added
Have gene markers so recombinant/ transformed bacteria can be recognised
Circular so stable
Explain why DNA is heated in the first step of PCR
To separate the two DNA strands
By breaking hydrogen bonds between bases
So that bases are exposed
To produce template strands for complementary copying
Explain why primers are added in PCR
Primer binds to DNA by complementary base pairing
It attaches to a specific section of DNA
DNA polymerase only attaches to double-stranded DNA
Primers reduce re-annealing(binding) of separated strands
Why is Taq polymerase used in PCR
It synthesises complementary DNA strands
Taq polymerase is heat stable
So does not be added again for each cycle
Process is more efficient than normal DNA polymerase
Explain why there are usually more than 100 copies of mtDNA in a cell but only two copies of nuclear DNA
There are many mitochondria per cell but there is only one nucleus
Explain how in the process of genetic fingerprinting, gel electrophoresis is able to distinguish between the VNTRs that occur at the same loci of different individuals
VNTRs with more repeats are longer
Phosphate groups of DNA have a negative charge
Fragments of DNA are attracted to the anode
Shorter pieces move faster
Longer pieces are more impeded by the agarose gel
Describe how electrophoresis can be used in genetic fingerprinting
VNTR sequences are used for genetic fingerprinting because these regions of DNA are unique to an individual
DNA is extracted and the quantity of DNA is increased by PCR
DNA is fragmented by restriction enzymes and the fragments are loaded into wells in agarose gel
They are placed at the end near the cathode
A direct current is applied and because the phosphate group of the DNA has a slight negative charge, the negatively charged DNA will move towards the anode
Shorter pieces of DNA move faster through the gel, moving further in a given time
Fluorescent dye is added to the and UV light can be used to identify the banding pattern after gel electrophoresis
Describe the role of insulin in the regulation of blood glucose concentration
Increases CELLULAR uptake of glucose from the blood by liver and muscle cells
This leads to increased respiration of glucose.
Insulin also causes the conversion of glucose to glycogen
