adaptors, probes, and blots

double digest

DNA sample is simultaneously digested with two different RE (cleave palindromic sequence).

used for cloning, DNA mapping, or restriction fragment length polymorphism (RFLP) analysis.

double digestion conditions

1. enzyme selection: 2 diff RE with compatible conditions like buffer pH and temp.

2. simultaneous digest for cutting

3. fragment analysis: electrophoresis

why do inserts have extra nucleotides before being inserted within the vector?

prevention of the frameshifting effect that would cause the ATG (methionine) not to be read as a start codon and instead as another amino acid.

Therefore, forward oligonucleotides must have RE sequences followed by two more nucleotides before starting with the gene.

For the reverse oligonucleotide, either add two extra nucleotides to re-establish the reading frame with the vector or add a stop codon to the oligonucleotide.

strong promotor

high transcription levels (eg. trp promoter)

This specificity can be advantageous for targeting gene expression to specific tissues or under specific environmental conditions.

CMV- mammal

Inducible promoter

on/off switch

Leaky system: lac promoter, can be induced with IPTG (IPTG is added, it binds to the lac repressor protein). but leaky due to transcribing without (basal).

Tighter control: T7 phage promoter. (eg. pET15b  vector)

Expression tags

facilitate the production and purification of proteins.

These tags are small polypeptide sequences that can be fused to the target protein, aiding in its purification without interfering with its biological activity. Once the protein is purified, the tag can be removed using proteolytic cleavage, leaving behind the desired protein in its native form.

How is T7 promotor used with E.coli RNA polymerase?

T7 promotor, Needs T7 RNA polymerase which is in certain strains of E.coli chromosome. pET vector series

The T7 RNA Pol gene is fused downstream of the E.coli lac promoter.

thus, IPTG induces both

Glutathione-S-Transferase (GST)

glutathione-removed by Reduced glutathione/ thrombin (cleaves)

6x his

ni2+- removed by his imidizole

protein A

igg- removed low pH

Maltose Binding Protein (MBP)

amylose-removed maltose

strep tag 2

streptavidin- removed by biotin or derivatives

how to identify of protein expression

Assay for protein activity

Label the protein with radioactive compound (methionine)

Visualize on a gel before and after induction

Use antibody against the protein or the affinity tag to detect

radioactive protein labeling

Transcription/translation system (rabbit reticulocyte lysate, wheat germ lysate)

add DNA

add RNA polymerase (depending on the promoter in the vector)

add amino acids

add labeled amino acid (methionine)

allow transcription and translation to proceed

fractionate a sample of the reaction on a polyacrylamide gel

RE optimal condition

ionic strength

pH

detergent

divalent cations, Mg++ (EDTA can inhibit enzyme reactions)

buffers (Tris-HCl vs Tris-acetate)

what occurs with incompatible REs

star activity→ cut wrong sequence

dcm

methylates 2nd cytosine

which RE bypass dam

bamhi

which enzyme is blocked my dam

bcii

What if we need to digest the DNA with an enzyme that is sensitive to dam or dcm methylation?

Use Dam-/Dcm-Methylase-Deficient Strains

By transforming your plasmid into mutant strains (dcm and/or dam genes), you can ensure that the DNA remains unmethylated and can be digested with enzymes that are sensitive to dam or dcm methylation.

sequential digest

1. Digest with one of the enzymes, repurify the DNA, then digest with the other enzyme.

2. Digest with the enzyme requiring low ionic strength first, heat inactivate (why? avoid carry over-star activity), add second buffer and second enzyme.

Conversion of 5’-overhangs and 3’-overhangs to blunt ends

3'→5' exonuclease and 5'→3' polymerase activities of T4 DNA Polymerase

DNA polymerase 1 domains

1. polymerase

2. 3’-5’ exonuclease

3. 5’-3’ exonuclease

Kienow fragment

polymerase and 3’to 5’ exonuclease

fills in receding 3’ ends to make blunt ends. by its polymerase

3’-5’ exonuclease activity of Klenow fragment removes protruding 3’ ends.

Adaptors

short, blunt-ended oligonucleotide that contain a unique restriction site

What is the requirement for selecting an adaptor for a particular sequence?

Must have RE  but the same RE must not be in the gene

Southern blot and purpose

DNA

transferring digested DNA onto a solid support (membrane) and detecting a specific fragment with a probe.

Purpose:

•Monitor the presence of a gene.

•Look for multi-copy genes.

•Look for homologues; inter or intra species.

•Look for chromosomal deletions.

southern transfer method

Digest DNA samples

Fractionate on an agarose gel

Transfer onto membrane by capillary flow aided by a transfer buffer

Hybridize probe

Scan/detect

Analyze results

Northern blot

RNA

Western blot

protein

influence of fragment size on southern transfer-digest

fragments of ~1kb are transferred quantitatively within 1 hour

fragments of ~15kb require at least 18 hours, non-quantitative

Fractionate on an agarose gel achieves what?

DNA separation by size (large to small)

why are the fragments transferred to a membrane in southern blot

DNA is bound to the surface of the membrane where it is now exposed (i.e. we now have easy access to the DNA fragments). The membrane is usually positively charged.

How does the membrane transfer in southern blot work?

Transfer buffer contains NaOH to denature DNA and facilitate its transfer to the membrane

Need a continuous flow of buffer, therefore the “wet paper” should be immersed in a pool of buffer (ie. wick effect)

prob of the southern transfer

sequence of DNA with an reporter sttached

• radioactive P

• enzyme that produces chemi-luminescence from a substrate molecule

• fluorescent tag

works by H bonding to complement and unbound are washed away. leaving bound reporters for detection

how do fluorescent probs bind?

they the primary antibody

how do fluorescent probe emit light and what are the 3 stages?

upon absorbing light energy, can reach an excited state, and then emit light energy. The three-stage process of excitation, excited lifetime, and emission

what are the 2 methods of End-labeling with polynucleotide kinase?

• forward RXN

• exchange RXN

what is an exchange RXN?

• the single stranded DNA/ RNA has a phosphate group

• excess ADP is added

• this causes PNK to convert ADP to ATP. Using the the phosphate on the single stranded DNA/ RNA .

• if the ATP is labelled (p-radioactive)

• LOW EFFIENCNCY RXN B/c the PNK can use the radio labelled p or the one attached to the strand.

what is an forward RXN?

• y-phosphate of ATP is transferred to the 5’end

• single stranded DNA/ RNA has NO phosphate group

• uses ATP and PNK, byproduct is ADP

• HIGHLY ACTIVE

why is a dephosphorylated strand important in forward RXN?

phosphatase to remove at 5’ end

Phosphatase prevents self-ligation

New strand is highly active

what is PNK catalyst?

transfers a phosphate from ATP to the 5' end of either DNA or RNA

How do we ensure that the 5’ end contains no phosphate?

• If using a PCR product, make sure the oligonucleotides are not phosphorylated

• dephosphorylate the ends with phosphatase

what is the hierarchy of PNK phosphorylation?

G>A/T>>C

How to do forward RXN when DNA is blunt or if the 5’ end is recessed (ie. 3’ overhang)?

higher concentrations of ATP m

and PEG* (polyethylene glycol). - crowding agent which increases the effective concentration of both the ligase and the DNA substrate in the tube.

southern transfer prob

The HexaLabel™ DNA Labeling Kit is designed for high specific activity labeling of DNA.

how does HexaLabel work?

• random primer method

• ie Random sequence primer are annealed to templet of denatured DNA

• complementary strands are synthesized with Klenow fragment

• with biotin-labelled supply of dNTP

• probes are hybridized

• membrane is washed

• dye treatment with (IR800) conjugated streptavidin b/c biotin was used

• streptavidin will bind to the biotinylated base

• signal

what is Stringency?

hybridization experiments to denote the degree of homology between the probe and the filter bound nucleic acid.

higher the stringency, the higher percent homology required between the probe and filter bound nucleic acid for detection.

high temperature and low salt influence on Stringency

more stringent as only perfectly matched hybrids will be stable

factors that can effect Stringency and hybridization

pH, temperature, probe length, detergent concentration, formamide concentration and salt concentrations

influence of temperature on probe

T_M (temperature at which 50% of the probe is bound)

influence of salt on probe

stabilizes nucleic acid duplexes

influence of formamide on probe

denatures DNA

^{rate of hybridization based on length is what}

^{the rate of renaturation of DNA in solution is proportional to the square root of the fragment length; thus, the maximal rate of hybridization is obtained with long probes.}

RFLP mapping steps

1.Isolate DNA from individuals

2.Digest with RE1, RE2, RE3, etc., in different tubes

3.Fractionate on a gel and perform Southern blotting

4.The sum of the patterns will be different if the individuals are not related.

partial digest

less enzyme and less time

Mapping DNA Fragments, libraries

northern blot

• RNA

• Not digested

• Denatured with glyoxal before electrophoresis

• Also commonly denatured with formaldehyde

•  Denatured with low concentrations (~0.05M) of NaOH for transfer

glyoxal benefits

• used in northern blot denaturation

• sharper bands

• less toxic than formaldehyde

what isn’t RNA, digested in northern blot? ie what does it allow to analyze?

• shows alternative splicing

• isoforms

• a specific probe to determine the tissue-specific or developmentally-regulated expression of the isoforms.

degenerate oligonucleotides

amino acids and many codons that include them

use the sequence range that has the least variability in nucleotide sequence. b/c less probs

western blotting

• proteins

• antibodies

applications of western blot

presence or absence, size, and abundance of target proteins in a sample which is beneficial for various scientific reasons across many fields of study. In this regard, the technique proves beneficial to evaluate the following: