Enzymes And Restriction Flashcards
What are 3 recombinant proteins that are made using genetic engineering?
→ Insulin
→ Interferon- involved in antiviral defence
→ G-CSF- promotes formation of bone marrow.
What is G-CSF?
→ produced by infected cells and induces antiviral defense
What do nucleases do?
→ Degrade nucleic acids by hydrolysing phosphodiester bonds
What does ribonuclease degrade?
→ RNA
What does deoxyribonuclease degrade?
→ degrades DNA
What do exonucleases degrade?
→ degrades from the end of the molecule
What do endonucleases degrade?
→ degrades from the middle of the molecule
What is the purpose of restriction?
→ to limit the transfer of nucleic acids from infecting phages into bacteria
What 2 things do restriction endonucleases do?
→Recognize a specific sequence
→ cut a specific sequence
What sequence does ECOR1 recognise?
→ GAATTC
What are the characteristics of recognition sites?
→ 4-8 base pairs in length
→ they are palindromic
How often does a 4 base sequence occur?
→ every 265 bases
When does a 6 base recognition sequence occur?
→ every 4096 base pairs
What 2 nucleases produce an overhang?
→ EcoR1
→ Kpn1
What nuclease does not produce an overhang?
→Alu 1
How are DNA fragments separated in electrophoresis?
→ by charge
Where does the DNA move during electrophoresis and why?
→ DNA is negatively charged due to the phosphate groups
→ when a current is applied the DNA moves to the +ve pole
What is the relationship between migration and fragment size?
→ Smaller fragments run quicker
How do you find out the size of the fragment?
→ You compare the digested fragments to the standard ones to compare the length
What does having two bands in a linear fragment mean?
→ it means you have two restriction sites
What kind of a mutation occurs in sickle cell?
→ A single point mutation
→ Instead of GAG you get GTG
What effect does the mutation have in sickle cell?
→ induces charges in the beta globin gene which makes it defective
What is the restriction site for the enzyme DDE1?
→ CTGAG
How do you test for sickle cell using DDE1?
→ purify DNA
→ Fragment of 450 bp
→ digest the PCR with DDE1
→ if there is normal beta globin - 2 restriction sites and 3 fragments
→ if there is sickle cell beta globin - 1 restriction site and 2 fragments
How can DNA molecules from different sourced joined together?
→ ECOR1 overhangs are compatible
→ Two fragments are mixed with DNA ligase and a recombinant molecule is produced
→ DNA ligase makes covalent phosphodiester bonds between DNA fragments
What does DNA polymerase do?
→ copies DNA based on a template
Why do you use DNA polymerase?
→ PCR amplification
→ Generations of probes
→ blunt ending of DNA overhangs
How does phosphatase work?
→ Hydrolyzes a phosphate group off its substrate
What two types of phosphatases are used?
→ Calf intestinal phosphatase
→ Shrimp alkaline phosphatase
Why do you use a phosphatase?
→ To prevent cut plasmids from resealing
→ If the phosphate group in the plasmid isn’t removed it can reseal
→ If it is removed then it cannot be resealed alone because DNA ligase needs a phosphatase group to work on one single size
What does polynucleotide kinase do?
→ Converts phosphate from ATP to substrate
→ Adds phosphate to the 5’ hydroxyl group of DNA or RNA
Why would you use a polynucleotide kinase?
→ To phosphorylate chemically synthesized DNA so that it can be ligated to another fragment
→ To sensitively label DNA so that is can be traced using radioactive or fluorescently labelled ATP
What is a probe?
→ fragment of ssDNA
→ 20-1000 bases in length
→ Complementary to the gene of interest
What are reverse transcriptases isolated from and why?
→ isolated from RNA containing retroviruses- HIV
→Not found in eukaryotes
What do reverse transcriptases do?
→ Synthesize DNA molecule complementary to a mRNA template using dNTPs
What is needed to form a DNA copy from RNA?
→ a primer
What is a random primer?
→ cDNA upto 700bp
→ covers all the length of the RNA molecules
What is an oligo dT primer?
→ useful for cloning cDNAs and cDNA libraries but some might not be full length
→ a TTT sequence is used because genes that code for proteins are polyadenylated
Why are specific primers designed for reverse transcriptase?
→ reverse transcriptase has a size limit
What does palindromic mean?
reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand
Why do bacteria produce restriction enzymes?
→work as defence system for bacteria from bacteriophage.
→The restriction enzymes cleave the phage DNA
Why do restriction enzymes not cleave their own DNA?
because of methylation of bacteria DNA so not recognised by restriction enzymes
How do phosphatases prevent resealing?
remove 5’-phosphate groups from DNA, RNA and both ribo and deoxy-ribonucleoside triphosphates
Blunt ends or sticky ends, which has a lower efficacy ligation?
blunt ends
In SCA, what restriction site is lost?
DdeI site (5’CTNAG3’)
