5. Chromosome Banding II

100X

100X

1000X oil

A chromosome

spread

Specific Stains for DNA

• Schiff’s or Feulgen

• Geimsa

To use Schiff’s reagent to stain DNA in a tissue, the tissue is first treated by mild acid hydrolysis, 1 M HCl, 60º C, 15 min.

Depurination by mild acid hydrolysis yields the aldo sugar 2-deoxyribose with afree viscinal hydroxyl. The ring can open with a free aldehyde group exposed.

Two aldehydes in close proximity will react with Schiff’s reagent to yield dark red stained DNA.

Onion root tip cells stained with Schiff’s and counterstained with Fast Green. The red is DNA. This root tip has been squashed.

I

A M

P

The three components of Geimsa stain.

What would make Geimsa specific for DNA?

What is this?

Speculate on the

mechanism of

Geimsa binding

to DNA.

Ethidium, proflavin and acridine orange are all fluorescentDNA stains.

Do you recall the mechanism of staining?

12

3

4

5

67

Do you recognize this structure?

3.4 Å

The Double Helix

side view

right-handed

antiparallel strands

20 Å

Top View

One strandis blue.

One strand is red.

The hydrogen bonds formed by A-T & G-C base pairing account for the two

strands fitting together, but they do not

account for the double helix being a stable structure.

van der WaalsBonding

Single van der Waals interactionsare 2-4 kJ/mol.

2-4 kJ/mole is very little energy, but when a molecule made of many atoms approaches another molecule of many atoms, the total energy can be substantial.

Ring structures are not necessarily flat. Consider glucose in the chair or the boat conformation.But what about DNA bases? Are they flat?

Watson-Crick Base Pairing

10.8 Å

11.1 Å

In the Watson-Crick model, the base pairs lie in the same plane, or close to it.Tilt of base normal to helical axis = -1.2°Average propeller twist = +16°

What is the distance betweenbase pairs in DNA?

C-GG-C

-61.0

C-GA-T

-44.0

C-GT-A

-41.0

G-CC-G

-40.5

G-CG-C

-34.6

G-CA-T

-28.4

T-AA-T

-27.5

G-CT-A

-27.5

A-TA-T

-22.5

A-TT-A

-16.0

Stacked Dimer Stacking Energy (kJ/mol)

Stacking Energies for theten possible dimers inB-DNA

The mechanism is intercalation. The ethidium slips in between the stacked bases of double-stranded DNA.

See the resemblance?

By what mechanism does methylene blue stain dsDNA?

• This is eosin, another common histological stain.

• Would eosin be a specific stain for dsDNA?

• Hint: Look at the charged groups.

Uses of Karyotyping

• Identification of specific chromosomes.

• Diagnosis of specific diseases.

• Prenatal diagnosis

FISH: fluorescence in situ hybridization

A fluorescent probe specific for a muscle protein gene binds to the two copies of the gene (yellow spots).

Using G-banding, one could identify the chromosomethat carries the muscle protein gene.

Chromosome PaintingSpectral Karyotyping

http://carolguze.com/images/chromosomes/sky3.gif

Each chromosome is hybridized with probes specific to sequences found on that chromosome.

This karyotype shows a translocation. The q arm of chromosome 22 has beenattached to the q arm of chromosome 9.

source: Stine, G.J., The New Human Genetics, 1989, Wm. C. Brown p 354

This 9/22

translocation is

indicative of

chronic

myelogenous

leukemia.

Number and type

of chromosomal

abnormalities

among spontaneous

abortions and live

births.

Griffiths et al. Intro. to Genetic Analysis5th ed. table 9-2

Experiment: Chromosome Banding II

• 1. Before lab, the slides with cells affixed will have been "aged" by incubation in a 60°C oven overnight.

• 2. Place slides in a solution of dilute trypsin and agitate gently for 15 seconds.

• 3. Rinse slide thoroughly in 0.01 M phosphate buffer, pH 6.8.

• 4. Stain in Giemsa stain for 5 min.

• 5. Rinse the slide twice in 0.01 M phosphate buffer, pH 6.8 and blot around the spread cells. Be careful not to blot off any cells. Allow the slide to dry on a hot plate for 5 minutes.

• 6. To mount, place one drop of permount off the glass applicator rod on the center of the spread cells. Lay a cover slip down over the drop and allow the cover slip to settle by its own weight until the permount has spread under the entire cover slip. If there is excess permount oozing from under the cover slip, scrape it off with a razor blade and place the slide in the fume hood until the permount dries.

Experiment: Chromosome Banding II (cont’ed)

• 7. Look for mitotic figures using 100x covering the area where the drop spread in search pattern. Use bright field.

– You can see the area by a faint purple stain.

– When you see mitotic figures, i.e. a group of chromosomes, center the group in the field and switch to the 1000X with oil.

• 8. The location of banded chromosomes can be recorded by using the X and Y vernier scales on the side and rear of the microscope stage.

• Please photograph your banded chromosomes using our Canon cameras.

Banded

Adam Shelly’sSectionWed. Spring 20101000X bright field

Scontras & Schwarz Eric Johnson’s section Thurs. 315 Spring 2010 1000X bright field

Susan Shao’s section 306 Tues, spring 2009

1000X, bright field

Kaitlin’s lab 3.28.13

Tara’s section