Section 5 - Recombinant DNA Technology and Genomics Flashcards
Describe the tools of recombinant DNA technology and explain how each functions: Restriction Enzymes
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Why is E. Coli often used to express YFP?
- Grow quickly and inexpensively
- Genetic engineering is simple
- Multicopy plasmids and strong promoters can drive expression
- Protein extracts are easily made
In order to express our human gene in E.coli, what barriers need to be considered?
(Use concepts from gene expression in prokaryotes and eukaryotes to outline the strategy and decision processes used to clone a gene de novo in E. coli)
- Eukaryotic genes have introns, E.coli can’t process these. So must use cDNA (derived from mRNA by reverse transcriptase enzyme, in order to have a DNA with no introns, only exons.)
- Human and E. coli promoter structures are different (euk=monocis; prok=polycis)
- They differ in their mechanism of translational initiation (euk=scanning; prok=recruiting via operators?)
- Different post-translational modifications occur in prokaryotic and eukaryotic cells …explore this
Because of the differences in translation, plasmid containing bacterial promoter, ribosome binding site (!!) – around the ATG site? of translation for translation of gene, and space for YFG to be inserted.
Put YFG right in between both cut sites, and now it’s ready to be expressed
In order to express our human gene in E.coli, what is the first barrier?
Eukaryotic genes have introns, E.coli can’t process these. So must use cDNA (derived from mRNA by reverse transcriptase enzyme, in order to have a ds DNA with no introns, only exons.)
Describe the tools of recombinant DNA technology and explain how each functions: Nucleic Acid (DNA) Hybridization (aka. DNA renaturation or annealing)— reversible melting
Purpose?
When is it performed?
Conditions?
Types?
(In case study: to find a tissue that expresses a lot of YFG - will detect YFG in a tissue (because not all tissues have same expression).) Searches for nucleic acids with a complementary sequence to our sequence of interest.
- the complementary strands of DNA reform the double helix, driven by the re-formation of the H bonds btwn base pairs.
- conditions: salt + slowly cooling down
- any 2 nucleic acid strands will hybridize if they share sufficient complementarity (ssDNA-ssDNA or ssDNA-RNA, or RNA-RNA)
- Hybridizations are often done after transferring the nucleic acid to a membrane.
Southern Blot: DNA probe to DNA on membrane (would use to see fi YFG is expressed in different organisms)
Northern Blot: DNA probe to RNA on membrane (to see in diff tissues)
Microarrays: DNA to DNA on glass slides. (uses mRNA to cDNA) (used for profiling RNA in different cell types–but not used often anymore) (ex, the cancer cell vs normal cell in same well seeing what gene is being upregulated, downregulated or unchanged. The wells will show whatever colour of probe is more prevalent.)
Explain Northern Blotting
Goal: to identify which tissue expresses YFG
Step 1: Isolate RNA from different tissues (lyse cells, then purify mRNA)
Step 2: Use agarose gel electrophoresis to separate the RNA on the basis of size (now you see the RNAs of different tissues)
- you’ll see 2 bands: bottom band for rRNA– so abundant that you still see them even after purifying for mRNA only
Step 3: Transfer the separated RNA to a membrane (nitrocellulose or nylon)– BLOTTING, 24hrs
- salt solution from sponge goes up into gel, picks up separated RNA fragments and gets blotted onto membrane, then paper towels absorbs salt solution
Step 4: Prepare a radiolabeled hot probe (=synthetic oligosaccharide) for fragment of interest. If it is dsDNA, the probe denatures it first. If ssDNA or RNA the probe must be the complementary strand.
-only need small fragment of YFG to use as probe
Step 5: Incubate the HOT probe with the filter (~10C below Tm) for 24hrs
- complementary strands will anneal
Step 6: Wash away the nonspecifically bound probe
- probes that didn’t find a match will not stick
Step 7: Expose the probed filter to X-Ray film to determine where (which tissue) the radioactive probe has hybridized (will show a band on film) –aka, where YFG is expressed
Explain Northern Blotting
Goal: to identify which tissue expresses YFG
Step 1: Isolate RNA from different tissues (lyse cells, then purify mRNA)
Step 2: Use agarose gel electrophoresis to separate the RNA on the basis of size (now you see the RNAs of different tissues)
- you’ll see 2 bands: bottom band for rRNA– so abundant that you still see them even after purifying for mRNA only
Step 3: Transfer the separated RNA to a membrane (nitrocellulose or nylon)– BLOTTING, 24hrs
- salt solution from sponge goes up into gel, picks up separated RNA fragments and gets blotted onto membrane, then paper towels absorbs salt solution
Step 4: Prepare a radiolabeled hot probe for fragment of interest. If it is dsDNA, the probe denatures it first. If ssDNA or RNA the probe must be the complementary strand.
-only need small fragment of YFG to use as probe
Step 5: Incubate the HOT probe with the filter (~10C below Tm) for 24hrs
- complementary strands will anneal
Step 6: Wash away the nonspecifically bound probe
- probes that didn’t find a match will not stick
Step 7: Expose the probed filter to X-Ray film to determine where (which tissue) the radioactive probe has hybridized (will show a band on film) –aka, where YFG is expressed
Describe the tools of recombinant DNA technology and explain how each functions: cDNA synthesis
- cDNA lack introns, thus good for expressing proteins, unlike genomic DNA. IS SINGLE-STRANDED!!
- use enzyme reverse transcriptase to make a complementary copy of DNA using RNA as template
- reverse transcriptase needs primer: poly-dT (synthetic oligosaccharide maybe?) will anneal to polyA tail of mRNA, then RT + dNTPs will make cDNA
- using for PCR, so don’t need to synthesize other strand, but if you wanted to make it, have to bring in DNA Pol
- treat with alkali to degrade RNA
Describe the tools of recombinant DNA technology and explain how each functions: Polymerase Chain Reaction
-Exponential amplification of any DNA from a source in which it is found as little as once (so that we can clone), by repeated extension of 2 primers.–purifying from other genes at same time
Required reagents: Template (ss or ds)DNA; 2 oligonucleotide primers which flank YFG on upstream on downstream and synthesize towards gene; dNTPs; DNA Pol
Step 1: Heat to separate strands.
Denature dsDNA template with heat (94C)
Step 2: Cool to anneal primers.
Lower temp to allow 2 primers (complementary to YFG) to anneal/hybridize to correct strand at 50C (below Tm). You must have sequence information to make the primers. One on each strand on opposite sides.
Step 3: DNA Synthesis.
Extend the primers with DNA polymerase +the 4 dNTPs and incubate at 70C so that DNA can be synthesized
Step 4: Repeat steps 1-3 ~30 times
-end up with 2^n DNA copies, n=# of cycles
Describe the tools of recombinant DNA technology and explain how each functions: Gel Electrophoresis
-DNA is sieved through pores of polysaccharide agarose gel, being pulled by an electric field, migrating toward + pole (bc - phosphate backbone). To separate different DNA fragments by size
- small DNA molecules move more easily through, and thus can migrate faster
-but charge doesn’t matter in terms of how fast/far it goes bc DNA has a uniform mass:charge ratio – separation only based on size
Detecting DNA in gel:
-stain DNA with EtBr (ethidium bromide) to make it fluoresce red in UV light
-if DNA amount is too low, must use Autoradiography: radioactively label the 5’ ends of DNA fragments using the enzyme polynucleotide kinase and gamma 32P-ATP. Now can be seen after exposure of gel to X-Ray film
-there’s also a buffer that keeps the pH neutral (salt)
2 Key advances in PCR
- Discovery of thermostable DNA Pols called taq polymerases.
They don’t denature easy– stable at high temps. Retrieved from thermophiles in hot springs - Thermocyclers that oscillate btwn the 3 required temps (50-94-70), now can do 30cycles/matter of hours
Describe the tools of recombinant DNA technology and explain how each functions: Restriction Enzymes
+DNA Ligase
(FIRST STEP IN CLONING)
used for creating recombinant molecules (we’re putting YFG in plasmid), to cut DNA into defined workable units
- usually use Type 2 enzymes, because are site-specific DNA binding proteins – and cut palindromic sequences=same on one strand as on the other (2-fold symmetry)
- can have 5’ overhangs, 3’ overhangs or blunt ends (go see pics, contrary to what you’d think)
- the same enzyme will cut the plasmid and YFG(uses PCR primer to attach to YFG)
Nomenclature: ex: HindIII is the 3rd RE isolated from Haemophilus influenza strain D
-Restriction Enzymes have 2-fold symmetry
DNA Ligase (isolated from T4 bacteriophages) is an enzyme that acts as glue, resealing sticky ends. Less efficiently reseals/ligates blunt ends. Uses hydrolysis of ATP as energy source. (Technically just seals the nicks, the ends re-anneal on their own bc of base pairing)
LIGASE REQUIRES PHOSPHATES AT 5’ END! otherwise no sealing
NOW we have a complete recombinant DNA plasmid molecule
Define and explain plasmids
3 Key features required to make plasmid useful
A plasmid is a fragment of DNA that replicates independently from the host chromosome
1. ORI (but YFG site can’t be here), so that it can replicate in a bacterial cell independently of the bacterial chromosome
2. A selectable marker: to detect plasmid once it’s in.
usually a gene encoding resistance to an antibiotic, Eg. Ampicillin
3. A site(s) into which YFG can be inserted. (aka, cleavage sites so that it can be easily opened to insert DNA fragment)
Describe the tools needed for gene cloning and explain how each functions.
We now have a DNA copy of YFG and are ready to clone it into the plasmid.
Need:
- restriction enzyme that’ll give sticky ends (to cut same site in plasmid and YFG)
- genome of bacteria in bacterial cell
-100ng of YFG
- thousands of ligated plasmids (‘vector’) (some will take up)
- ATP
- T4 DNA Ligase
-Transformation provided by cell
- DNA Pol, oligo primer, dNTPs, ddNTPs for DNA sequencing
Describe the tools of recombinant DNA technology and explain how each functions: DNA Sequencing
-allows us to make sure taq polymerase didn’t make a mistake.. so checks base by base.
Uses ddNTP (have no 3’ OH so no strand extension)
-DIDEOXY/CHAIN TERMINATION/SANGER SEQUENCING
- the ss YFG DNA fragment is hybridized with a fluorescent DNA primer (which is complementary to first 4 nucs of YFG)
- DNA Pol and a whole bunch of dNTPs are added to 4 separate tubes
- each tube receives a small amount of one Chain-Terminating ddNTP (one gets A, one T, one C, one G)
- each time a dNTP (eg, dA) is supposed to add that letter to the sequence, a ddNTP (eg, ddA) goes in its place, and that’s one DNA copy (all the DNA copies are from same sequence, and all have same 5’ end, just terminate at diff points and so differ by 3’ end)
- contents of tubes put in 4 diff wells in gel E. and so the bands in each lane represent fragments that have terminated at a given nucleotide, but at diff positions in the DNA
- by reading the bands in order from bottom(5’) to top (so longest strands at top), you get the sequence of the newly synthesized strand –COMPLEMENTARY TO OUR YFG
Now novel methods have been devised to obtain large amounts of DNA sequence at minimal cost (automated using reading of wavelengths of fluorescent dye on ddNTPs)
