Gene Technologies Flashcards
What probe do you use for a recessive disease
Probe complementary to the recessive faulty allele
Need 2
What probe do you use for a dominant disease
Probe complementary to the dominant faulty allele
Only need 1
Why can protein structure be used to determine evolutionary relationship
Closely related means similar base sequence so similar primary structure of amino acids and therefore protein structure
Why is it better to compare DNA base sequence than protein structure
Degeneracy of triplet code
Longer so more specific
DNA base sequence contains introns but protein structure doesn’t
Why is it better for restriction endonuclease to be complementary to TATCTGTCTAT than just TAT
TAT repeated too often
Longer one less likely to be complementary to wrong parts
Enzyme is complementary to a lot of DNA for TAT so produces more small fragments
Why can plants synthesise insect protein
Genetic code is universal The same bases exist in all organisms Same triplet codes for same amino acid Desired gene can be transcribed into mRNA And translates into insect protein
What does recombinant DNA technology allow
Genes to be manipulated, altered and transcribed from organism to organism
Better understanding of how organisms work
Design new industrial processes and develop medical applications
3 important features of the genetic code
Universal: Same triplet codes for same amino acid
Degenerative: More than one base/triplet for each amino acid
Non-Overlapping: Each base only part of one triplet/read once by ribosome
5 steps for producing and conducting gene transfer and cloning
Isolation Insertion Transformation Identification Cloning
Benefit of DNA being universal
All organisms have the same triplets coding for same amino acid
So transferred DNA can be transcribed and translates
During protein synthesis
3 methods of isolation
Conversion of mRNA to cDNA using reverse transcriptase
Restriction endonucleased to cut a fragment with desired gene
Create gene in gene machine
How is reverse transcriptase used in isolation
mRNA mixed with free DNA nucleotides
Enzyme reverse transcriptase added and mixed in
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing via reverse transcriptase
Into cDNA
Degrade mRNA with RNAase/strong alkali to leave cDNA (single stranded)
Added DNA nucleotides and DNA polymerase to make cDNA double stranded
Advantage of using reverse transcriptase in isolation
mRNA in cytoplasm already been spliced so all introns removed
mRNA easier to obtain
Use mRNA base sequence to work out sequence of exon bases without the need for splicing
If being actively expressed, cytoplasm will contain lots of mRNA (e.g insulin mRNA abundant in beta cells of pancreas)
Cells only contain 2 alleles for a gene
Bacteria don’t have enzymes to deal with introns
Use of restriction enzymes in isolation
Cut/hydrolyse phosphodiester bonds in DNA
At specific recognition sequences complementary to their active site
These are palindromic sequences (base pair reads same both ways)
If target gene has recognition sequence before and after
DNA incubated with specific restriction endonuclease
Same enzyme must be used for DNA of interest and plasmid so sticky ends match and can form complementary base pairs via H bonds
Where do restriction endonucleases cut
Specific recognition sequence complementary to active site
These are palindromic sequences
Read the same in opposite way
What two things do restriction endonucleases produce
Sticky ends
Blunt ends
What are sticky ends
Transformation
Is Sam amazing
Yes
What are sticky ends
Overhand and complementary
Same restriction endonucleases
Needed in transformation and modification
What are blunt end uses
PCR
Gel electrophoresis
Restriction mapping
Regulatory genes
Gene that regulates the expression of one or more structural genes
By controlling the production if a protein
Which regulate their rate of transcription
Proteome
Entire set of proteins that is or can be expressed by a genome, cell, tissue or organism at a certain time
Gel electrophoresis
Method for separation and analysis or DNA, RNA and protein and their fragments based on their size and charge
Oligonucleotide
Small overlapping single strands of nucleotides that are assembled into the desired gene
Advantage of gene machine
Easily transcribed and translated by prokaryotes as they have no introns in their DNA
Explain how the gene machine works
Desired nucleotide sequence inputted into computer
Synthesis of oligonucleotides
Which are overlapped
And joined together and made double stranded by PCR
Gene is inserted into bacterial plasmid and cloned
Sequenced, those with errors rejected
Vector
DNA carrier (bacterial plasmid/virus) used to transfer foreign DNA into cells
How is gene inserted into vector
Use same restriction endonucleoase to isolate DNA fragment and cut open DNA vector
So that it has a promoter and a terminator
So RNA polymerase can bind before the gene of interest and release after it
Producing complementary sticky ends
Target DNA fragment anneals to vector DNA by complementary base pairing between the sticky ends
DNA ligase uses to join DNA fragment and vector DNA at the sugar phosphate backbone (ligation)
Phosphodiester bonds formed
New recombinant DNA
Two vectors
Plasmid
Bacteriophage/virus
Two ways to encourage DNA vector to be transferred into host
Giving bacteria a short electrical shock
Opens temporary small pores in the cell membrane
Calcium phosphate makes temporary pores in cell membrane
Method for in vivo cloning
Cut desired gene from DNA desired organism
Using restriction endonuclease
Use mRNA from cell of organism
Reverse transcriptase from desired DNA
Make artifical DNA with correct base sequences Using DNA polymerase Cut plasmid open With same restriction endonuclease Producing stick ends DNA ligase used to join Return plasmid to bacterial cell
Why must organisms be identified
Not all vectors take up target DNA and become recombinant
Not all host cells become transformed by taking up recombinant vectors
Only transformed host will synthesis desired protein
Only want to clone transformed cells
Marker gene
Allows easy identification of cells that have taken up a genetically transformed plasmid
Those that have not will not be fluorescent/identifiable
3 ways to use marker genes
Antibiotic resistance
Fluorescence
Enzyme markers
How do you identify colonies that have taken up recombinant DNA with replica painting/stamp
Incubate on a growth medium
Sterile velvet surface used as a stamp
To transfer cells from each colony onto replica plates
One has the normal medium but one has the condition which only those transformed can survive in
Clone those successful
Explain the outcomes for replica painting/stamp
Some cells haven’t taken up plasmid
So killed by both antibiotics
Some cells have taken up original plasmid
So resistant to both types
Some taken up transformed plasmid
So resistant to one but not the other since the gene is cut and disrupted by inserting foreign DNA
Primer
Short pieces of single stranded DNA
Complementary base sequence to bases at the start of DNA fragment you want to isolate
Prevent strands sticking back together
Allow DNA polymerase to attach and join free nucleotides together
Purpose of PCR
Used to amplify a single DNA fragment into millions of copies
Stages are automatically repeated many times
Number of DNA molecules doubles every cycle
So rapid and efficient
PCR
Polymerase Chain Reaction
3 steps for PCR
Separation
Annealing
Synthesis
Why is the PCR cooled
Primer won’t attach at 95 so H bonds can form
Steps for PCR
Heat DNA to 95 degrees which breaks weak hydrogen bonds and separates strands
Add primers and nucleotides
Cool to 50 degrees to allow binding of nucleotides and primers
Add thermostable DNA polymerase (Taq)
Heat to 75 degrees
DNA polymerase joins nucleotides together by phosphodiester bonds
Repeat cycle many times
Amplify DNA
Number of DNA molecules after each PCR
2^n
n=number of cycles
Number of cycles in PCR
Log2(n)
n=number of DNA molecules
Advantage of using a thermostable enzyme in PCR
Enzyme is stable even at high temperatures
So not denatured at high temperatures
Making it efficient at amplifying DNA
Disadvantages of in vivo cloning
Slower - One DNA replication per cell division so limited by growth of cell
Cant be used to copy partly broken down DNA
Not very sensitive so need a large DNA sample to start with
Need to isolate DNA fragment from DNA before it can be inserted into host
Advantages of in vivo cloning
Almost no risk of contamination
Cells have mechanisms for correcting errors made when copying genes so much more accurate
Can be used to clone large DNA fragments
Can be used to produce protein or mRNA from the inserted DNA as well as target DNA
Disadvantages of in vitro cloning
Can only be used to copy DNA
Lacks error-correcting mechanisms so error rate higher
Cloning becomes unreliable when used to copy DNA fragments longer than 1000 base pairs
Advantages of in vitro cloning
Rapid - can produce millions of copies of DNA in hours
Only replicates target DNA so no need to isolate DNA fragment from DNA
Can be used to copy partly broken down DNA
Very sensitive, only requires a small sample of DNA to start with
Three uses/benefits of recombinant DNA tech
Agriculture; GE for high yield, more nutrients, pest resistance, drought and temperature resistant
Industry; GE organisms to make large quantities of enzymes quickly and cheaply for industrial processes
Medicine; GE organisms can be produced to make large quantities of drugs and vaccines quickly and cheaply
Issues of recombinant gene technologies
Antibiotic resistance marker genes within GE could lead to transfer of these genes to pathogens and decrease antibiotic effectiveness
Introducing herbicide resistance genes could result in transfer to wild species when they interbreed and produce weeds resistant to herbicides
Why are viruses or liposomes used as vectors
Plasmids cannot carry DNA into human cells
Liposome
Lipid droplet like a vesicle that carries DNA into cell
Fuses with cell surface membrane and releases DNA into cell
DNA doesn’t move into nucleus so daughter cell won’t have functional gene
Somatic vs germline gene therapy
Somatic is DNA transfer to normal body tissue
Germline is DNA transfer to cells that produce eggs or sperm
Somatic gene therapy restricted to patient and isn’t inheritable
VNTR’s
Variable Number Tandem Repeats
Not the same as introns since not in the gene
Describe how genetic fingerprinting is carries out
DNA extracted from sample
Cut into segments using restriction endonucleases
Leaving VNTR’s intact
DNA fragments separated using gel electrophoresis
Mixture put into wells on gel and electric current passed through
Immerse gel in alkaline solution/two strands separated
Southern blotting
DNA fixed to nylon/membrane using UV light
Radioactive probe added which is picked up by required fragments
Identified using X-ray film/autoradiography
