Molecular - Blots

Question 1

Southern Blot Steps

Answer 1

1. Digest DNA (REs); 2. Separate (electrophoresis); 3. Transfer to solid matrix; 4. Hybridize. 5 Expose Xray film to filter

Question 2

Transfer set up

Answer 2

Plastic support. Membrane dips into buffer. Place gel. Filter paper. Stack of paper towel/sponge. If

Question 3

Restriction Enzymes

Answer 3

Type 1 - random cut sites (circa 1960). Type 2 - specific cut sites (1970). Host protected by methylation; cut pallindromic sequence (same on both strands)

Question 4

Restriction Enzymes examples

Answer 4

EcoR1 - 5 g aattc 3; BamH1 - g gatcc; usually 4 or 6. 7 is possible, usually has a variable in the middle

Question 5

How often do restriction enzymes cut?

Answer 5

(1/4)^n. n is number of bases in cut site. Can be influenced by base pair bias (sometimes G/C more rare)

Question 6

Restriction mapping

Answer 6

Compare digests and double digests. Set up relative cut sites.

Question 7

Separation. Based on?

Answer 7

Gel electrophoresis. Based on charge (DNA/RNA are -ve), size (gel matrix), SDS???

Question 8

Agarose

Answer 8

not very precise. For sequencing (small as 30 bp), need polyacrylamide (4%)

Question 9

Protein separation

Answer 9

much more differences, different sizes. SDS to denature. need gradient gel (4% agarose near top, 20 ish at bottom to stretch out small ones)

Question 10

Hybridization

Answer 10

Probe hybridizes at lower temp and higher [salt]/detergent. Put in drum, uses much less solution.

Question 11

Hybridization variables

Answer 11

Too low = probe binds too much, inappropriately. Higher [salt] = more stable double forms, probe binds stronger (if lowered, it is called stepped down). Increase detergent = pulls off some probe (can be fully stripped to restart)

Question 12

Crosslinking in Southern blot

Answer 12

UV cross links DNA to nitrocellulose; some don’t need it, but it won’t hurt

Question 13

Radioactive detection

Answer 13

32P or 33P (longer 1/2 life) for X ray film; probe can be clone. Kind of messy/dangerous

Question 14

Chemiluminescent detection

Answer 14

relies on biotin (Vit. B), co-factor binds tight to protein; probe can be clone

Question 15

Probe types

Answer 15

1. Synthetic oligo. (15-30); weaker signal. 2. PCR. Strong. 3. Clones, strong; insert into plasmid, grow culture, purify plasmid, cut out again, gel (separate clone and plasmid), then can cut out that section

Question 16

Label a probe

Answer 16

Enzyme adds a radioactive phosphorus in a phosphate group (enzyme exchanges P31 for P32)

Question 17

Label your chemiluminescent probe

Answer 17

Add psoralen molecule to biotin with carbon linker. Psoralen (3 ring structure kinda like base pairs) intercalates at T residues. Then UV drives covalent crosslink of psoralen - biotin attached to any probe

Question 18

After making biotinilated probe…

Answer 18

Probe hybridizes, then wash with streptabidin, then add alkaline phosphatase which removes phosphate from PP dioxitane which emits light

Question 19

Streptabidin

Answer 19

Bacterial protein which binds almost irreversibly to biotin. Used to tag a biotin probe

Question 20

Phototope star

Answer 20

By NEBL. Releases light in response to certain enzyme activity. Alkaline phosphatase goes to biotin; it removes (P) from proprietary agent (PP dioxitane); this intermediate emits light at 461nm, darkens Xray film.

Question 21

Phototope star and other chemiluminescent issues

Answer 21

Working blind in washing steps. Can’t tell if too much too little until you put in cassette. If it didn’t work you have to restart at hybriziding.

Question 22

Congenital adrenal hyperplasia

Answer 22

in CYP21 (21 hydroxylase). Also a pseudogene version (CYP21P). Digest region. Both have same Taq1 cut sites. People with CAH lack a polymorphism so it doesn’t get cut there and one spot is empty; Pseudogene confirms a negative result.

Question 23

Southern Hybridization

Answer 23

Determined a protein sequence, so you make a probe. Challenging because of redundancy in genome. Unknowns: Y = pyrimidine, R = purine. N = any nucleotide. Create a degenerate primer = mixture

Question 24

Making primer

Answer 24

1st nucleotide bound to column, nucleotide which is protected at 3’ so only one binds, rinse, deprotect, add next one (protected), rinse, deprotect, etc.

Question 25

Making degenerate primer

Answer 25

For degenerate primer, same process, but for degenerate nucleotide positions, add a mixture of the unknowns. Creates many different primers, at least one of which will hybridize.

Question 26

Degenerate primer tips

Answer 26

Generally avoid locations with 4 nucleotide degeneracy, number of primers starts to increase exponentially; good targets are strong acidic and strong bases (important in structure and are conserved), and amino acids with only 1 codon.

Question 27

RNase and Northern Blots

Answer 27

One of most stable enzymes ever; many RNases on fingers. Makes work with RNA difficult! Therefore materials need to be clean always. Treat with CEPC to stop its activity. (CEPC can be stopped with autoclaving).

Question 28

Difference b/w Northern and Southern

Answer 28

Don’t need a digest for Northern; different splicing for RNA can make 2 bands;

Question 29

Challenge with RNA in Northern Blot

Answer 29

Single strand can fold on itself - add formaldehyde which denatures RNA and keeps it linear

Question 30

Western Blot and protein detection summary

Answer 30

Electrophoresis, Transfer (diffusion not good - 2nd electrical force in other direction to force it out of gel), Detection (antibodies)

Question 31

Antibodies structure

Answer 31

Heavy chain: 200+ Variable regions, 20+ Diversity regions, 6 J; V and J in light chains also; all linked to a C region; some slippage can occur too. Results in millions of antibodies. Each B cell makes one type.

Question 32

Antibodies (AB) in Western

Answer 32

Add a mouse AB which binds to protein of choice; then limited digestion to recover mouse C region and inject it into a rabbit; purify rabbit anti-mouse AB which will bind to C region of mouse AB.

Question 33

Visualizing in Western Blot

Answer 33

Rabbit anti-mouse is bound to a reporter enzyme. Blocker used to block the rest of membrane so rabbit AB only binds to mouse AB. Add chromogenic substrate, visualize.