DNA HYBRIDIZATION TECHNIQUES AND PROBES

Hybridization

Formation of hydrogen bonds between complementary bases

Hybridization

It is a process of establishing noncovalent and sequence specific interaction between two or more complementary strands of nucleic acid into a single hybrid

DNA PROBE / GENE PROBE

are synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing.

Probe

is also an oligonucleotide [short sequences of DNA]

Primer

is usually used for amplification – for PCR – initiates the DNA synthesis [didikit sa side ng target = initiate a formation of new strand]

Probe

is a short sequence of DNA that directly binds to your target

Northern Blot

Southern Blot

Western Blot

3 MAJOR HYBRIDIZATION TECHNIQUES TO BE USED:

DNA

is a very long strand, composing different sequences of nucleotide; it is a chain or polymer of nucleotide

DNA

is portioned in a way that for each portion, there’s a specific gene.

Northern Blot

detects RNA

Southern Blot

Detects DNA

Western Blot

for proteins; used as a confirmatory test for proteins [i.e. HIV]

Far Eastern Blot

Detects Lipids

Southern Blot

Named after Edwin Southern [the one who developed the technique]

Southern Blot

Can analyze any gene or gene region in prokaryotes or eukaryotes at the molecular level.

Southern Blot

Uses labelled PROBE - The probe will not bind if the DNA is double stranded; it needs to be single-stranded to form hybridization

Southern Blot

is helpful in detecting specific sequences of DNA

1. Restriction Enzyme Cutting and Resolution

2. Preparation of Resolved DNA for Blotting (Transfer)

3. Blotting (Transfer)

Procedure for southern blot

3 hours or more

how many hours is the digestion of DNA using Restriction Enzymes?

Type 2 endonucleases or Type 2 Restriction Enzyme

Cut the DNA into specific size using __________ – cuts in the middle

gel electrophoresis

once cut, we have to separate the fragments or nucleic acids using _____?

10-50 μg of high-quality (intact) genomic

_______ DNA is used for each restriction enzyme digestion for Southern analysis

longer runs at low voltage

Large fragments requires _______ to get the best resolution in southern analysis

restriction enzyme activity was incomplete

Large aggregate [single band] on top indicates:

DNA is degraded

Smear in the lower region indicates:

Large fragments (>500BP)

______ are more efficiently denatured if they are depurinated before denaturation

DEPURINATION

Uses Hydrogen Chloride [acidic] solution which loosen up the larger fragments by removing purine bases from the S-P backbone

Hydrogen Chloride [acidic] solution

this solution loosen up the larger fragments by removing purine bases from the S-P backbone

DENATURATION

If it’s less than 500 base pairs, you can perform _____ directly

(NaOH) – Sodium Hydroxide

DNA is denatured by exposing the gel to a strong base ______ is a strong alkaline solution

Denaturation

Promotes breakage of the hydrogen bonds holding the DNA strands to one another

Denaturation

forming ssDNA

Nitrocellulose

[preferred as compared to nylon]

Apurinic Site

if the purine is removed, it is now called?

Purine

we remove this so that the denaturant will have more access to destroy the hydrogen bonds for easier denaturation.

Adenine and Guanine

examples of purine

Polyvinyl difluoride (PVDF)

used for immobilization of proteins

Antibodies

are proteins – they are immunoglobulins

Single-stranded DNA

avidly binds to nitrocellulose membranes with a non-covalent, but irreversible, connection

Nitrocellulose

is the most versatile medium for molecular transfer applications

1. Capillary transfer

2. Electrophoretic Transfer

3. Vacuum Transfer

Three types of Transfer Method

ssDNA

much preferred than dsDNA in membrane transfer

Transfer Method

Move the DNA from the gel to a membrane substrate for probing

CAPILLARY TRANSFER

The only manual method out of the three; unlike electrophoretic and the vacuum – requires sophisticated procedures

Capillary Transfer

is the simplest because it is based only on how the molecules move in a solution

Capillary Transfer

Simple and inexpensive; no instruments required

Capillary Transfer

Slow and prone to staining artifacts

Capillary Transfer

The filter paper is submerged on the buffer. It will absorb the buffer, and at the same time there’s a stack of absorbent paper on the top. When there’s absorbent paper, as long as there’s moisture, it will absorb it. The DNA will go along from the agarose gel to the membrane

ELECTROPHORETIC TRANSFER

Semi-automated; we don’t rely on the capillary movement of molecules, but rather in the charge of the molecules

ELECTROPHORETIC TRANSFER

Uses electric current to move the DNA from the gel to the membrane

ELECTROPHORETIC TRANSFER

The current carries the DNA transversely from the gel to the membrane

Semidry Transfer

The filter paper is the only one submerged in the buffer

VACUUM TRANSFER

Relies on the pressure

Vacuum Transfer

It is submerged completely in the buffer, and there’s a hole on the bottom

Vacuum Transfer

Uses suction to move the DNA from the gel to the membrane in a recirculating buffer

Vacuum Transfer

it avoids discontinuous transfer due to air trapped between the membrane and the gel

• Vacuum Oven (80°C, 30 to 60 mins)

• UV Cross linking

After transfer, the DNA can be permanently immobilized to the membrane through:

NORTHERN BLOT

Sample: Total RNA/ mRNA

Northern Blot

Designed to investigate RNA structure and quantity

NORTHERN BLOT

Uses approximately 30 μg total RNA or 0.5 to 3.0 μg polyA

RNA

are applied to 0.8% to 1.5% agarose gel/polyacrylamide gels

inhibits binding of RNA to nitrocellulose

Denaturant must be removed from the gel by rinsing the gel in deionized water before transfer because it

Prehybridization

Prevent the probe from binding to nonspecific sites on the membrane surface

Blocking agents

block other parts of the DNA for the probe to focus on the target

Denhardt solution

Ficoll, polyvinyl pyrrolidine, bovine serum albumin

Salmon Sperm

blocks other parts of the DNA

Formamide

is a sulfhydryl reagent that removes hydrogen bonds; so, if there’s hairpin structures, formamide with remove it.

0.01% SDS with formamideincubate

blocks in RNA

Prehybridization

incubate for 30 mins to several hours

PROBES

Single-stranded fragment of nucleic acid attached to a signal- producing moiety

PROBES

Hybridize specifically with the target DNA or RNA

PROBES

Contain normal nitrogen bases that can hybridize with complementary DNA or RNA and resistant to nuclease degradation due to:

Probe length

range from tens to thousands of base pairs

Probe

is highly specific; should be complementary to the target for it to bind.

Probe

will not be destroyed by the nucleases as it resist denaturation through its structure

PROBES

resist degradation because we remove the charges [it is uncharged]

1. Peptide nucleic acid

2. Locked nucleic acid

3. Unlocked Nucleic Acid

OTHER NUCLEIC ACID PROBE TYPES

Other Nucleic acid Probe Types

Synthesized using chemical methods

Other Nucleic Acid Probe Types

Resistant to nucleases that degrade DNA and RNA

Peptide Nucleic Acid

• Most commonly used

• resist degradation

Peptide Nucleic Acid

Uncharged nucleic acid; removed the negative charges by removing the phosphate [unphosphorylated]

Locked Nucleic Acid

It will become accessible to the nucleases because the 2nd and 4th carbon of the sugar will have a phosphodiester bond in between

Unlocked Nucleic Acid

The 2nd carbon and 3rd carbon is joined together, remove the bond between the two

Locked Nucleic Acid

• Negatively charged

• Synthesized like to DNA/RNA

Locked Nucleic Acid

• Easy to make LNA-DNA chimeras

• Strand invasion not yet reported

• Soluble like DNA/RNA

Locked Nucleic Acid

LNA-DNA can activate RNase H

Cellular delivery with lipid

Low toxicity with animals

Peptide Nucleic acid

• Uncharged

• Synthesized like peptide

Peptide Nucleic Acid

• Easy to make PNA-peptide

• Outstanding strand invasion

Peptide Nucleic Acid

• Solubility varies with sequence

• No activation of RNase H

Peptide Nucleic acid

• Cellular delivery with lipid

• low toxicity in animals

1. Direct

2. Indirect

Types of Probe Labeling

Direct Probe Labeling

- We use radioisotopes [old method]

- Expose to radiation to create an activity

Indirect Probe Labeling

Non-radioactive; biotin or digoxigenin

Probe Labeling

For visualization of bound probe to the target fragments in the membrane

Probe labeling with 32P

Classic Method in probe labeling

Probe Labeling

Based on indirect detection of a tagged nucleotide incorporated in or added to the probe

1. End labelling

2. Nick Translation

3. Random Priming

METHODS IN DNA PROBE LABELLING

End Labelling

Terminal transferases or T4 polynucleotide kinase

End Labelling

In this method we will transfer the substance going to the 5’ end