Y2: Gene technology Flashcards
What are the three methods to make/produce DNA fragments?
- mRNA to make copy with reverse transcriptase
- Restriction endonucleases
- Gene machine
What is the method for using reverse transcriptase to copy the insulin gene?
(Using reverse transcriptase to make DNA fragment (gene))
5
- Beta cells from the islets of langerhans produce insulin mRNA
- Reverse transcriptase uses DNA bases to make a single stranded complementary DNA (cDNA)
- cDNA separated from mRNA by hydrolysis with an enzyme
- DNA polymerase forms a second strand from the cDNA
- This forms a full copy of the human insulin gene
What is the method for using restriction endonucleases to make DNA fragments?
2
Blunt ends
- Restriction endonuclease has a recognition site for the DNA sequence it cuts at this site is not palindrome
- This produces a straight cut so blunt ends
What are restriction endonucleases?
2
Enzymes that hydrolyse DNA
Cut DNA a known sequences
What is the method for using restriction endonucelases to make DNA fragments?
2
Sticky ends
- Restriction endonuclease has recognition site for DNA sequence that is in a palindrome sequence
- Cuts and produces a staggered cut so makes sticky ends
What are the two types of DNA fragment restriction endonucleases make?
Blunt ended
Sticky ended
How are DNA fragments produced using the gene machine?
4
extra
1
- Use bioinformatics to determine base sequence of gene
- Computer designs series of oligonucleotides and they are assembled
- The oligonucleotides are then assembled to form a gene
- Replicated by polymerase chain reaction
(5. Using sticky ends it can inserted into plasmid (vector))
What are the advantages of using the gene machine?
3
- Any sequence can be produced in a short time
- Has great accuracy
- Free of introns so can be transcribed and translated by prokaryotic cells
What are the advantages of in vitro gene cloning?
2
- Extremely rapid so is valuable when there is only a small bit of DNA
- Does not require living cells
What is a disadvantages of in vitro gene cloning?
As it copies it rapidly if there is contamination that also multiplies by loads
What is used in the polymerase chain reaction?
5
- DNA fragment to be copied
- DNA polymerase
- Primers
- Nucleotides
- Thermocycler
What is the method for the polymerase chain reaction?
1. Separation of the DNA strand
2
- DNA fragments and primers are put into a vessel and into a thermocylcer to vary the temperature over time
- Temperature is then increased to 95*C causing the H bonds between the DNA strands to break separating the DNA strands
What is the method for the polymerase chain reaction?
2. Addition of primers
3
- The mixture is cooled to 55*C causing the primers to join/anneal to their complementary bases at the end of the DNA fragment
- Primers attach to provide starting sequences for DNA polymerase to begin copying from and prevents the strands from rejoining
- DNA polymerase is added
What is the method for the polymerase chain reaction?
3. Synthesis of DNA
3
- Temperature is cooled to 72*C, this is the optimum temperature for DNA polymerase
- DNA polymerase adds nucleotides along the separated DNA strands beginning at primer and ending at end
- It catalyses the formation of phosphodiester bonds
What does in vitro mean?
Using test tube/scientific equipment to make something
What does in vivo mean?
In organism
Why are sticky ends important?
3
- The ends of the fragment have unpaired bases
- The two ends are complementary to each other
- DNA ligase forms phosphodiester bonds between nucleotides once complementary bases have lined up to create one piece of combinant DNA
What are the issues with using sticky ends?
3
- Plasmid sticky ends can stick to each other not the gene that you are trying to insert
- Sticky ends of the gene can stick to each other creating a loop not associated with the plasmid
- RNA polymerase and transcriptional factors need promoter region on DNA to recognise where to start transcription
In Vivo Cloning? How is the DNA inserted into the host cell by a vector?
4
- Use a vector eg: plasmid
- Plasmids usually carry genes for antibiotic resistance (you need to know which ones)
- Cut the plasmid with the same restriction endonuclease as to make the DNA fragments so you get complementary ends
- Mix the fragments with the cut plasmids and DNA ligase
In Vivo Cloning: How are the plasmids inserted into the bacteria?
3
- Mix the plasmids and the bacteria in a medium containing Ca2+
- Modify the temperature
- Some bacteria will have taken up the plasmids and some of the plasmids will have the incorporated gene (transgemic plasmids)
In Vivo Cloning: how do you identify which plasmids have taken up the gene?
method 1
2
method 2
2
- Fluorescent markers: insert gene for fluorescence into plasmid,
- If it is fluorescent there is no gene as the gene is inserted into the fluorescence gene and it is still functional
- Enzyme markers: lactase as it turns colourless substrate blue
- I f it is blue there is no gene as the lactase is still functional so gene is not inserted
How does DNA hybridisation work? (DNA probes)
5
- Take a sample of the DNA to be tested
- Heat it up to 95*C to break H bonds
- Mix sample and DNA probe together
- Cool it down so the probe can attach to the sample DNA - allows H bonds to form
- Detect to see if probe has bound to DNA
- Wash off solution from fixed surface
- Detect
What is a DNA probe?
2
Short, single stranded length of DNA with a label attached
-Can be a fluorescent molecule or radioactive nucleotide
What are the advantages of genetic screening?
2
You can test for specific alleles so:
1. can do personalised medicine
2. genetic counselling with experts to discuss risks and decisions
Eg: about having children or treatments etc.
