Chapter 5 - Analysis and Characterization of Nucleic Acids and Proteins

Restriction Enzyme

Endonuclease activity at a 4-6bp recognition site. Splice at their recognition site to separate DNA into 2 pieces.

Restriction Map

DNA is exposed to different restriction enzymes separately, and then in different combinations. Can determine rough fragment order based on the cuts/sites.

Restriction Mapping Steps

1. Add enzyme one and analyze fragment sizes

2. Add enzyme two to uncut DNA and analyze

3. Repeat as needed

4. Add in combinations

5. Using what you know build a rough map

Star Activity

Restriction enzymes cleave at the improper site under suboptimal conditions. Otherwise they’re very specific for sites they cleave.

Restriction Fragment Length Polymorphisms (RFLPs)

The number and location of restriction sites for a given restriction enzyme are not the same from person to person. The same enzymes can produce different fragments of size and number.

CRISPR

A restriction system in archae and gram negative/positive bacteria that uses RNA components to find cleavage sites. Useful for both DNA and RNA expression manipulation.

CRISPR Acronym

Clustered Regularly Interspaced Short Palindromic Repeats

CRISPR Structure

Repeat sequences that are separated by spacers matching the genomic regions of previous infected material (bacteriophage, plasmids, etc).

DNA from new invaders is incorporated as a short repeated sequence, serving as adaptive immunity.

CRISPR Sequence of Events

1. When an invasion is present, short RNA transcribed from the spacer regions (crRNA).

2. crRNA forms a complex with tracrRNA (a trans-activating factor) and Cas enzyme.

3. crRNA homology leads crRNA to its target where it binds and cuts the invading motif.

Cas

Enzyme required for CRISPR system to work.

Cas requires a specific PAM on crRNA to cut the DNA. The PAM varies from species to species and is part of the sequence complimentary to the target.

PAM facilitated the RNA:DNA hybrid between the target and the crRNA.

Types of CRISPR/Cas

Type I: Target dsDNA

Type II: Target dsDNA

Type III: Target ssDNA and RNA

CRISPR/Cas in Research

Used to target specific genomic sequences of choice. Provides the specificity of restriction enzymes but with more versatility.

The DNA will then be repaired by homologous recombination, using a synthetic donor that can provide whatever sequence change you want.

CRISPR Gene Regulation

Can lead activators or repressors (in place of Cas9) to gene promoter sites to regulate transcription.

Southern Blot Summary

• Target: DNA

• Probe: Nucleic acid

• Purpose: Gene structure

Northern Blot Summary

• Target: RNA

• Probe: Nucleic acid

• Purpose: Transcript structure, processing, gene expression

Western Blot Summary

• Target: Protein

• Probe: Protein

• Purpose: Protein processing, gene expression

Southwestern Blot Summary

• Target: Protein

• Probe: DNA

• Purpose: DNA-binding proteins, gene regulation

Eastern Blot Summary

• Target: Protein

• Probe: Protein

• Purpose: Modification of western blot using enzymatic detection (PathHunter), also detection of specific agriculturally important proteins

Far-Eastern Blot Summary

• Target: Lipids

• Probe: None

• Purpose: Transfer of high-performance liquid chromatography (HPLC)-separated lipids to polyvinyl difluoride (PVDF) membranes for analysis by spectrophotometry.

Nitrocellulose

The solid support used for blotting techniques. Tightly binds the target material.

Southern Blot Procedure

1. Digest DNA with restriction enzymes (varies based on purpose - 50ug of DNA per enzyme needed) and appropriate buffer, ~3 hours or more.

2. Run on gel electrophoresis and observe. Should see a smear representing DNA of almost every size, and equal intensity across lanes.

   • Too much at start = too little restriction enzyme activity

   • Too much at end = DNA is degraded

3. The dsDNA on the gel is soaked in weak HCl to de-purinate the DNA and make denaturing easier. NaOH is then used to denature the dsDNA.

4. ssDNA is transferred to a solid substrate, usually nitrocellulose. The ssDNA binds with a noncovalent and irreversible connection. Target is negatively charged and membrane is positively charged.

5. Probe hybridization.

Nitrocellulose Membrane Specifications

• Binds 70 to 150ug nucleic acid per square centimeter

• Pores are suitable for a few hundred to 20,000 bp in length

• High binding capacity for protein and nucleic acid

• Most versatile medium for transfer

Transfer Methods

• Capillary transfer

• Electrophoretic transfer

• Vacuum transfer

Capillary Transfer

The gel is placed on top of a reservoir of buffer with filter papers on top and on bottom. Capillary action brings the buffer through the bottom filters, passed the membrane and into the top filters, pulling DNA with it to the membrane.

Electrophoretic Transfer

Uses electrodes. The cathode (-) is attached to the gel, and the anode (+) the membrane, carrying the DNA from the gel to the membrane.

Vacuum Transfer

The gel is placed on top of the membrane, and a vacuum is applied underneath. Pulls the DNA from the gel to the membrane.

Prehybridization

Involves incubating the membrane in the same buffer in which the probe will be added into.

Northern Blot Purpose

• Investigate levels of gene expression and stability

• Investigate structural RNA anomalies from synthesis/processing, such as from alternative splicing

Can help determine/reveal mutations, overall

Northern Blot Procedure

1. Isolate RNA, quantify (30ug OR 3ug polyA) and add to an agarose gel (0.8 to 1.5%).

2. Gel electrophoresis is carried out to determine transcript size. Denaturation occurs during electrophoresis and a separate step is not needed.

3. Lanes are cut from the gel and soaked with ammonium acetate to remove the denaturant, and stained to assess quality.

4. Probe hybridization.

Western Blot Purpose

Resolves protein by either molecular weight or charge

Western Blot Steps

1. Isolate protein (usually from serum, cell lysate, or extract), quantify (need 50ug), and denature (Tris HCl or SDS usually).

2. Load onto polyacrylamide gel (SDS-PAGE) and run with standardized ladder.

3. Blotted to membranes, usually Nitrocellulose, by capillary or electrophoretic transfer.

4. Probe hybridization with antibodies and gel is washed with the same buffer.

5. Incubates for 12 to 16 hours, then washed again in the same buffer. Substrate is added for color/fluorescence.

Epitopes

Antigenic sites on proteins where antibodies bind

Protein Blotting Membrane Types

• Nitrocellulose (best - easily treated to prevent antibody from binding the membrane instead of the protein)

• PVDF

• Anion (DEAE) or Cation (CM) Exchange Cellulose

Southern/Northern Blot Probes

A single stranded nucleic acid attached to a signal producing moiety.

• Peptide Nucleic Acids (PNA)

• Locked Nucleic Acids

Contain normal nitrogenous bases, but a different type of backbone. Resistant to nuclease degradation and hybridize to the target more easily.

Western Blot Probes

Specific binding proteins or antibodies.

• Primary Antibody - binds directly to the protein of interest

• Secondary Antibody - used for visualization of the protein band of interest

DNA Probes - Short vs Long

Longer probes are more specific, requiring more base pair matches, while shorter probes can produce higher background signals due to more non-target complimentary DNA.

RNA Probes vs DNA Probes

RNA probes are often more sensitive since the RNA/DNA hybrid forms a MUCH stronger helix than DNA/DNA.

Both types of probes, however, have approximately equal binding affinity.

RNA probes are often less stable and incorporate radioactive nucleotides to increase sensitivity.

Monoclonal Antibodies

Bind to a specific epitome

Polyclonal Antibodies

Recognize multiple epitomes

Hybridomas

Hybrid cells that grow in culture and secrete antibodies