Bio. 551 Rec. DNA

Dr. Michelle Mardahl

Exam II. 37.5 points:

Define the function of 6 of the following and list procedures/applications. Example: oligo d(T) - binds polyA RNA used to isolate mRNA prior used to create cDNA (3 points) Grade the 1^st 6 answered.

Broad Host range Plasmid- Plasmids that can reside in many types of unrelated bacterial strains. Used as vectors to deliver genes to bacteria that are resistant to conventional transformation protocols. Applied extensively for industrial use and plant genetic engineering

Triparental mating- Mobilization of a plasmid into a recipient cell using 3 bacterial strains to participate in the mating mixture. One strain contains a self-transmissible plasmid, another has a target plasmid that needs to be transferred into the recipient strain which is needed for the cloning applications. Even if the two plasmids are members of the same inc. group they will coexist long enough for the mobilization to occur. Useful for cloning, transposon mutagenesis.

Conjugation- gene transfer among prokaryotes which is mediated by a conjugation plasmid which facilitates transfer of a plamid via pilus. Efficient method of gene transfer in strains in which transformation is inefficient.

Post segregational killing- Ensure the stability of broad host range plasmids. The kilA and kil E genes present in broad host range plasmids prevent daughter cells from surviving unless BHRP is transferred because they code for a long lived toxin and a short lived antitoxin respectively.

Incompatibility groups- plasmids groups that are established on the basis of genetic stability of genetic markers. Plasmids within an incompatibility group can not co-reside in the same cell without selective pressure.

Rnase H- Removal of the RNA template during cDNA synthesis reaction, nicks RNA/DNA hybrids, provides primers for second strand cDNA synthesis

RACE- Rapid amplification of cDNA ends used for to obtain full length cDNA clone for each mRNA including 5' untranslated leader sequence and 3' ends.

RPA- RNAse Protection Assay- Hybridize single stranded probe + RNA sample; Incubate w/ RNase; Run out on a gel, bands on gel used to quantitate of amount of target RNA.

Transduction- is the transfer of genes from bacteria via a phage (prokaryotes) or with eukaryotic cellular sequences via retroviruses.

List the advantages/disadvantages of 2 types of transformation protocols used in bacteria (2 points)- the two types discussed in class are:

1. Chemical Methods- E.coli cells take up purified DNA in the presence of cold calcium chloride mixed with DNA and then subjected to brief heat treatment.

2. Electroporation. Exposure to a strong electrical pulse causes a transient increase in the permeability of cells that allows DNA to cross the cell envelope. Efficiency depends on the strength & duration of pulse. Tradeoff of increase permeability with decreased cell viability- 25-50% of the cells die during the pulse. Cells need to be extensively washed to reduce ionic strenght and concentrate the cell suspension.

advantages disadvantages

1. Heat Shock Rapid, easy, cheap Inversely proportional to DNA size; SC DNA works best

2. Electroporation Very efficient; TE= 10⁶ - 10¹² More expensive, need cuvettes & electroporator

Linear efficiency over a

1. Lipid-mediated or Lipofection- Cationic & neutral lipid vesicles (liposomes) bind DNA (presumably via charge interactions). Mechanism of entry is not understood. May bind via negatively charged sialic acid moieties on cell membrane prior to internalization

2. Inorganic Calcium Phosphate- DNA ppt. adheres to cell surface then internalized.

3. DEAE dextran- Positively charged polymers- DNA ppt. adheres to cell surface then internalized

4. Electroporation - electric pulse causes transient pores. Optimize parameters (voltage & duration) and pH changes during/after the pulse (=cell death) [ use BES vs. HEPES buffered salines]for each suspension cell type. For stable lines grow 2 generations (48 H) in nonselective media and then transfer to selective media. Need many more cells (5X then Chemical Delivery). DNA integrated 1- several copies. DNA size is not limiting.

5. Receptor Mediated. Requires noncovalent conjugation of the DNA with a ligand.

As per notes all methods except DEAE-dextran mediated are good for stable expression

Stable transfection- DNA integrates into the chromosome permanently. Requires cell lines and a selectable marker such as G418 resistance gene. Advantageous for this application because a recombinant protein needs to be purified from transfected cells by large scale requires continuous expression

Negative Controls- 1 or 2 dishes of cells transfect with a carrier DNA/buffer used to dilute plasmid or gene. Cells should not become rounded and glassy in appearance. For stable expression- no colonies should be visible after growth with selective media

Transfect cells with a positive control to see relative toxicity of transfection (use GFP construct to quantitate the # of fluorescent cells via fluorescence microscopy)

1. Bake all glassware, spatulas, stir bars etc. at 200 C, 4-6 hours

2. Autoclave all tips, tubes

3. Designate chemicals for RNA work (limited use); Designate electrophoretic equiptment never exposed to RNAse; Use gloves when touching or weighing out.

4. Treat all water/solutions with 1% diethylpyrocarbonate (DEPC)- then autoclave

5. Clean pH electrode w/ DEPC water CsCl can be baked at 200 C

6. Care w/ Tris buffers- make up with DEPC treated water that has already been autoclaved

7. Wear gloves at all time, change them if touch skin or surface that might have been touched by hands

8. Use sterile plastic pipets and tubes (ones that are individually wrapped are best)

9. Include denaturing reagents for RNAse (guanidium isothiocyante, SDS, Ranasis, Inhibitase, Vanayl ribonucleotides, phenol, proteinase K)

1% SDS; Tris H-Cl in DEPC treated water- Tris is to buffer the RNA so it does not degrade due to extreme pH; SDS denatures Rnase and facilitates cell lysis by punching holes in the cell membrane

Equilibrated Phenol- homogenize, disrupts cell membrane & denatures RNase

Chloroform/ Isoamyl alcohol- homogenize= to prevent loss of poly A+ RNA; & stabilized interphase

Centrifuge. Draw a picture of the centrifuge tube and label the components and where RNA is supposed to be - top phase is aqueous phase- (RNA IS HERE); Interphase- RNAse/ Protein contaiminants Bottom Phase- Organic phase

1/3 volume 8 M LiCl- LiCl will precipitate large RNA and leave DNA and tRNA, & most polysaccaharides in solution

100% Ethanol- precipitate RNA

Full outline of protocol from notes:

1. Grind tissue in liquid nitrogen if necessary

2. Add 1% SDS, Tris-HCl pH 9.0. Equal volume equilibrated phenol. Homogenize

3. Add chloroform, homogenize again

4. Centrifuge to separate phases- take aqueous phase- proteins (RNase) are in the interphase- careful to not disturb

5. Re-extract aqueous phase 3x w/ phenol/chloroform

6. Ethanol ppt. and resuspend in DEPC water

7. Add 1/3 volume 8M LiCl and precipitate overnight at 4 C

LiCl will precipitate large RNA and leave DNA and tRNA, & most polysaccaharides in solution

8. Centrifuge, wash pellet in 2 M LiCl and ethanol precipitate again

Once you resuspend your RNA in 100 µl of __DEPC dH20___, you quantitate the amount by taking a spectrometric reading of a 1/100 dilution of RNA. Given that 1.0 A OD₂₆₀ of RNA = 40 µg/ml, calculate the yield and determine purity given (1 point)

OD₂₆₀ = 0.2 and OD _260/280 = 2.1- indicator that the RNA is pure (1.9-2.0) Lower A260/280 rations indicate DNA and or protein contamination

0.2 (OD260) x 40 µg RNA/ ml (OD260) x (1 ml/ 1000 µl) x (100) x 100 µl total sample = 100 µg

RNA is at 0.8 µg/µl Total Yield is 80 µg

1 µg/ 0.8 µg/µl = 1.25 µl

How much of your sample should you aliquot to run out 1 µg of total RNA on a gel?_____1.25 µl__(0.5 pt).

Explain how you isolate poly A RNA to construct cDNA (3 points) Describe by any of the following

A. Oligo dT Cellulose

1. 18-30 dT oligonucelotide attached to cellulose beads binds poly A tails of mRNA in total RNA

2. Bind in 0.5 M NaCl

3. Wash away non-polyA_ Rna in 0.1 M NaCl

4. Elute bound poly A+ RNA in no-salt buffer

5. Repeat entire cycle Yield 1-3% of total RNA. Check for ribosomal bands on agarose gel.

B. Poly-U paper

1. ~100 Us attached to paper discs binds binds poly A tails of mRNA in total RNA

2. Bind in high salt

3. Wash with medium salt

4. Elute bound poly A+ RNA in no-salt buffer

C. Magnetic Streptavidin spheres

1. Hybridize biotinylated 18-30 dT oligonucelotide to poly A tails of mRNA in total RNA

2. Add strepavidin (binds strongly to biotin) attached to magnetic beads

3. Pull beads (w/ polyA+RNA attached via biotin/oligo dT link) to the side of the tube with a powerful magnet

4. Wash the beads several times in high salt

5. Elute bound poly A+ RNA in low buffer

Total RNA- rRNAs (different sizes for different species but usually 18S (4.7 kb) & 28S (1.90 kb); also see 4-5S (0.10-0.15 kb- tRNAs & 5S rRNA) indicator if RNA is undegraded- should see sharp clear bands (note should not be present w/ polyA isolation). mRNAs (smear- all different sizes), snRNAs (small); Won't see tRNA here because won't ppt. w/ LiCl

Lane 1 total RNA Lane 2- Poly A RNA

rRNA 28s _____ smear w/ faint 28s/18s bands

rRNA 18s _____

rRNA 5s -------

1^st Strand Synthesis

mRNA + Oligod(T)XhoI +MMLV RT + dNTPs + CH3-dCTP

2^nd Strand Synthesis

Rnase H + DNA pol I. + dNTPs

Ligate EcoRI linkers

Or describe figure 6-7 a or b. p 98 from text

See pdf files from Mike Cleary's talk