Lecture 9 - Recombinant DNA And Molecular Cloning 1 Flashcards
What does recombinant DNA engineering provide the means to?
Assemble genetic information in new combinations in a directed way
What is a key recombinant DNA technique?
Gene cloning
How does gene cloning work?
Gene of interest linked to a plasmid vector which enables a,privation and propagation as a pure population of molecules in a cell
Why is molecular cloning of DNA important? 6 reasons
It purifies and amplifies individual fragments of DNA of genes of interest
Helps obtain their DNA sequences
Determine the gene structure and regulation
Perform site directed mutagenesis to investigate function
Express and purify protein for biochemical/structural analysis
Enable genome analysis by creating overlapping clones of genomic DNA
Enzymes for recombinant DNA engineering - why is type II restriction endonucleases used for?
Cleave DNAs at specific sequences
Enzymes for recombinant DNA engineering - why is type II methylate used for?
Methylates dnas at specific bases
Enzymes for recombinant DNA engineering - why is dna polymerase used for?
Copy dna form a primer at 3’ end
Enzymes for recombinant DNA engineering - what is rna polymerase used for?
Makes as rna copy of dna from a promoter
Enzymes for recombinant DNA engineering - what is reverse transcriptase used for?
Makes a dna copy of rna from a primer at the 3’ end
Enzymes for recombinant DNA engineering - what is DNA ligand used for?
Covalent lay joins two dna molecules or fragments
Enzymes for recombinant DNA engineering - what is exonucleases used for?
Remove nucleotide residues from the end of dna
Enzymes for recombinant DNA engineering - what is terminal transferase used for?
Adds a homoplmer tail to the end of dna
Enzymes for recombinant DNA engineering - what is polynucleotide kinase used for?
Add a phosphate to the 3’ end of dna
How can strains of bacteria become resistant to bacteriophage infection?
The appropriate endonucleases within the bacteria recognises specific sites in infecting dna and destroys it,
How can strains of bacteria that are resistant to bacteriophage infection protect their own dna from being cut if it is in the same sequence as infecting bacteria?
Expresses cognate methylates that modify dna at corresponding sequence specific sites in dna
What is the requirements for type II restriction endonucleases?
Magnesium and not ATP
restriction enzymes commonly recognises what type of sequences and example?
Symmetric palindromic. For example BamHI recognises G cut GATCC and binds to a homodimer forming a 2-fold symmetric enzyme-DNA complex
Do type II restriction endonucleases cut outside their recognition sequence to one side?
Yes
How many types of termini are produced by type 2 restriction enzymes?
3 - 3’ recessed ends, blunt ends and 5’ recessed ends
What does the 5’termini of each strand retain?
Phosphoryl group
What does the 3’ termini of each product retain?
Hydroxylated
What does EcoRI produce in terms of ends?
Staggered end
What type of end does EcoRV form?
Blunt
What can block restriction enzyme cleavage of DNA?
Methylation added by cognate methylates
What does dna ligase catalyse?
Formation of 5 - 3 prime phosphodiester binds in ds dna or to repair strand nicks and join adjacent Okazaki fragments
What is the ligase cofactor?
RATP for t4 DNA ligase
In what case can DNA ligase join two fragments together?

Ones with comparable sticky ends with a hydroxyl group at the free 3’ end and the phosphate group at the free 5’ end
Can DNA ligase also join restriction fragments with blunt ends?
Yes
What do high concentrations of DNA in lifetime lead to?
Inter-molecular ligation producing linear DNA
What do low concentrations of DNA in ligase lead to?
Infra-molecular ligation producing circular dna
What can ligase do to two restriction fragments with comparable ends?
Link them to give a covalently closed circular product at low concentration
What way does DNA polymerase synthesis DNA and uses what substrate?
5’ to 3’ using deoxyribonuclotide triphosohate as a substrate
Are DNA polymerase processive?
Yes
Many DNA polymerase have a 3’ to 5’ exonuclease activity what is this and what does it do?
Proof reading to eliminate errors in dna synthesis
What do some DNA polymerase have which removes DNA ahead of them?
5’ to 3’ exonuclease activity
What does klenow sub-fragments if DNA polymerase I lack?
5’- 3’exonuclease activity
What is 5’ to 3’ exonuclease activity used for?
Initiating DNA synthesis from oligonucleotide primers for radioactive labelling or chain terminator DNA sequencing
Why is T4 DNA polymerase useful for trimming restrictive enzymes fragments?
5’ to 3’synthesis activity and high level 3’-5’ exonuclease activity
What is used for chain terminator DNA sequences?
T7 DNA polymerase
What is reverse transcriptase used to make and how does it do this?
Complementary DNA (cDNA) from RNA templates initiating synthesis from an oligonucleotide primer
What thermosensitive polymerases are used for PCR?
Taq, Vent and Pfu
How can DNA polymerases be used to radioactively label restriction fragments?
By using an alpha labelled dNTPin the polymerase reaction
How does klenow DNA polymerse I used to make a radioactive DNA probe from the entire DNA fragment using random hexanucleotide?
Denature DNA and anneal hexanucleotide primers (hybridise randomly)
Synthesis of labelled dna strand
Denature dna and use probe
What does T4 Polynucleotide Kinase do?
catalyzes the transfer and exchange of phosphate from the g position of ATP to the 5’hydroxyl terminus of double-and single-stranded DNA and RNA
What is polynucleotide kinase used to label?
5’ end of restriction fragments or synthetic single strand oligonucleotide to use as probes using g-P32-ATP as the radioactive nucleotide; to phosphorylase PCR products made with non-phosphorylase primers ready for ligation
Where are cloning vectors derived from?
Plasmid, phage or a combination of both
How do Plasmid vectors work?
vector DNA introduced by transfection
How do phage vectors work?
vector DNA introduced by transduction (phage infection)
How does a combination of phage and plasmid vector work?
vector DNA introduced by transduction
What are the common properties of cloning vectors?
Unique restriction sites to facilitate cloning of insert DNA
genetic marker(s) to select for or identify cells containing the vector
Ability to promote autonomous replication and amplify from a single copy
minimal non-essential DNA to maximize size range of cloning
What are plasmid cloning vector features?
Selectable genetic marker – constitutively expressed gene encoding antibiotic drug resistance
A replication origin (ori)for autonomous replication in bacteria
• A unique restriction enzyme sites where DNA-restriction fragment
What is the process of molecular cloning? Preparing vector DNA
Prepare vector DNA by cutting at the unique restriction enzyme site in plasmid to produce a linear molecule
What is the process of molecular clone - step 2 purify?
Prepare and purify the DNA to be inserted by cutting with the same restriction enzyme or an enzyme that produces the same type of cohesive ends
What is the process of molecular cloning step 3 - mixing
Mix dna at low concentration and add ligase
Process of molecular cloning step 5 - transformation
Transformation into an E. coli strain with optimal genetic features for molecular cloning
What is the 2 ways e.coli cells can transform with lighted DNA within?
(a) Using CaCl2 - treated E. coli cells and heat shock
(b) Using high voltage electroporation
What cells survive on an antibiotic resistant place when selecting?
Only cells containing a plasmid survive but many may not have plasmids with an insert
What is used to prevent self-ligation of vector ends
Phosphatase
Why are modern plasmid vector designed?
engineered to replicate at high copy number, to be of minimal size with an artificial polylinker cloning region that contains multiple unique restriction sites, and the capability for rapid screening of recombinant s
What does Screening for recombinant plasmids by blue/whitecolony colour test rely on?
E.coli strain expressing an inactive version of beta-galactosidase
How is the activity of beta-galactosidase restored?
Association with an alpha peptide
How do you screen recombinant clones by nuclei acid hybridisation?
Transfer onto nylon filter,
Denature dna
Hybridise to prove
Wash and expose to X-ray film
CN ends produced by different restriction enzymes be comparable and if so give an example?
Yes, BamHI (5’G/GATCC3’) and BglII(5’A/GATCT3’) produce identical cohesive ends that can be efficiently ligated
How would you legate BamH1 and Bg/II?
Fill in the ends or resect DNA ends of vector and insert with T4DNA polymerase plus dNTPs to make them blunt and then perform blunt end ligation
Then Fill in ends of insert and ligate linkers containing recognitions sites of vector
How is Klenow DNA polymerase I or T4 DNA polmerase used to modify DNA-restriction fragments with recessed 3’ ends
Polymersiation
How is T4 DNA polymerase used to modify DNA restriction fragments with protruding 3’ ends?
3’-5’ exonuclease digestion and polymerisation
Can an EcoRI cut vector be blunt end lighted to a Sacl restriction fragment?
Yes
Linker ligation strategy - what drives addition of linkers by bling end ligation making it efficient?
very high concentration of the duplex phosphorylated linker molecule
Adapter ligation strategy - what is addition of adapters driven by?
Hugh concentration of the duplex phosphorylated linker molecule
Genetic variants of E. coli K-12 improve the efficacy and safety of recombinant DNA cloning - How?
Type I restriction modification systems can potentially dstroy foreign DNA-cloned in bacterial cells
Type I restriction modification systems can potentially dstroy foreign DNAcloned in bacterial cells - how is this done through ecoK restriction system?
Encodes proteins that efficiently degrade foreign DNA that is not properly methylated at the sequence 5’-AAC-(N) 5-GTGC-3‘, which would occur in many DNAs to be cloned
Why does the hsdR- strain allow propagation of plasmids with foreign DNA?
the gene encoding the Type I restriction enzyme R is deletedand the plasmid is not degraded. The methylase gene M is still retained.
What isn’t good for a successful gene cloning experiment?
Rearranged or deleted insert dna
Most e.coli k12 strains used for cloning has mutations where?
RecA gene which encodes an essentialgene for general homologous recombination to prevent spontaneousrearrangement between repeated sequences
How does recA enhance the bio safety of E.coli K12?
recAdeficient strains are sensitive to ultraviolet light
Some mutations cause loss of the gene encoding DNA specific endonuclease. What is this called and why is this good?
EndA. Loss of this endonuclease greatlyincreases plasmid DNA yields and improves the quality of DNA that isisolated using standard biochemical preparations
What does PCR obtain?
Pure dna in a test tube
What is used in PCR that is stable and active in the temperature changes seen in PCR?
Thermostable DNA polymerase
How does PCR work?
by amplification of a target DNA sequence defined by the specific hybridisation of oligonucleotide primers and their extension by DNA polymerase
PCR needs to knowledge of target sequence at the 3’ end of PCR primers need to be complementary to what?
Target sequences 17-25 nucleotides long
What is the synthesised product of copying reaction in each cycle used for?
a template in a subsequent reaction cycle
What does multiple cycles of PCR leave you with?
massively amplified population of predominantly identical double strand DNA molecules (an amplicon) with a length size representing the distance apart of the primer pair
PCR – the process in the initial cycles - how do you get the right length?
Anneal primer to DS DNA after denaturation.
Dna synthesis
Continue through many cycles until you get the correct length of DNA needed (this will occur due to the back and forth nature of this experiment)
How do you add nucleotide sequences onto the amplicon ends using PCR?
Add them to the end of the PCR primer and they will be copied in throughout
PCR can be used to amplify desired DNA with primers that contain appropriate restriction endonuclease sites in the 5’end. How does this happen?
Restriction site sequence in the 5’ extension is incorporated into the end of the double stranded PCR amplicon during the 2nd and 3rd round of PCR. Then amplicon is cut with appropriate restriction enzyme for each end to make cohesive ends necessary for ligation with a vector
What does PCR error rate depend on?
whether or not thermostable polymerase has a proof reading function (3’ to 5’ exonuclease activity)
What is important when considering what thermostable enzyme is required?
Error rate and processivity
What is required if PCR product is to be used for molecular cloning?
Low error rate
What does Long range PCR use?
Taq polymerase and a proof reading polymerase
