Lecture 2 | 09/08 Nucleic acids and tools for analysis

Forms of DNA

A-form and B-form: right-hand helix
Dehydration of DNA drives it into the A form
Z-form DNA is Purine and Pyrimidine repeat. Normally, it takes 10 repeats to form z-form.

Poll Challeng Question

Is more likely to form Cruciform.

The yellow highlighted part is the evidence.

DNA Secondary Structure

Triplex DNA

Hairpin and Cruciform Structures

G-quadruplex DNA

Seperating and Probing Nucleic Acids

Methods

• Ultracentrifugation (by density)
• Non-denaturing gel electrophoresis (by size and shape)
• Denaturing gel electrophoresis (by size)
• 2-D gel electrophoresis (by size, shape, and supercoiling)

Ultracentrifugation

Based on density.

Advantage

Get very pure samples

Disadvantage

Takes a long time

DNA can be seperated by different G-C percent.
G 151.13 g/mol
C 111.1 g/mol
A 135.13 g/mol
T 126.1133 g/mol

A little density difference

Example with yeast DNA:
3 distinct bands can be observed:

• Mitochondrial DNA (20% G-C)
• Chromosomal DNA (50% G-C)
• Ribosomal DNA (60% G-C)

Non-denaturing gel electrophoresis seperates nucleic acids

Based on shapes and size.

The Shape of DNA

The DNA fragments have the same size but different shape, the migrate in different speed.

Visualization

1. Using EtBr(Ethidium bromide ) to intercalating dyes to visualize nucleic acids under UV light.
   

2. Using radioactive labels and autoradiography.
   
   32P is fre

Denaturing gel electrophoresis

By size.

DNA Strands Must be Seperated to Identify Specific DNA Sequences.

1. Denaturation can be spontaneous or catalyzed by enzymes
2. AT-rich regions (arrows) spontaneously denature first
   1. Because there areonly two hydrogen bonds and C-G has 3.

DNA Denaturation can be measured using a Spectrophotometer

G-C Content and Salt Concentration can Affect DNA Denatuaration

Thesalt neutralizes the negative charges on the DNA backbone.

GC-rich and high salt -> highest Tm
AT-rich and low salt -> lowest Tm

Poll Question

Answer: Sample 2
Just look at that figure

Methods for Seperation/Identification of Nucleic Acids

Labeled probes to single-stranded nucleic acids

• Southern blot
• Northen blot
• In situ hybridization

Visualizing Specific Nucleic Acid Sequences: Southern and Northern Blotting

Used to determine

• Presence or absence
• Relative levels
• Orientation(DNA)

We just transfer gel to nitrocellulose because nitrocellulose is more stable than gel.

There is an assumption that these organs is producing approximately the same amount of GAPDH messenger RNA. That is why we can use it as a loading control.
So now we could say, there is more PECAM-1 RBA in the kidney, the lung and the trachea

Microarray: Quanitfy Multiple DNA and RNA Molecules

Can quantify multiple DNA and RNA molecules.

Why go back from RNA to DNA? Because DNAs are more stable than RNA in solutions.

Visualization DNA in cells/tissues: in situ hybridization

Sometimes we want to know where are specific nucleic acid sequences in a chromosome or in a cell.

Steps

1. Fix the cells/tissue in denaturing conditions
2. Incubate with a probe complementary to the sequence that you want to visualize
3. The probes can be radioactive or fluorescently labeled

Why this picture has four dots?

• We are in metaphase spreads. We are in mitosis.

Poll Question

If we only want to analysis the amount only one gene, we can juse use northern blot