5.

Chromosome Banding II

1000X oil

100X

100X

A chromosome
spread

Specific Stains for DNA

Schiffs or Feulgen
Geimsa

To use Schiffs reagent to stain DNA in a tissue, the tissue is first treated by mild
acid hydrolysis, 1 M HCl, 60 C, 15 min.
Depurination by mild acid hydrolysis yields the aldo sugar 2-deoxyribose with a
free viscinal hydroxyl. The ring can open with a free aldehyde group exposed.
Two aldehydes in close proximity will react with Schiffs reagent to yield dark red
stained DNA.

Onion root tip cells stained with Schiffs and

counterstained with Fast Green. The red
is DNA. This root tip has been squashed.

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 fluorescent

DNA stains.
Do you recall the mechanism of staining?

Do you recognize this structure?

2

TheDoubleHelix

20

sideview
righthanded
antiparallelstrands
3.4

TopView

Onestrandisred.

Onestrand
isblue.

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 Waals

Bonding

Single van der Waals interactions

are 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?

WatsonCrickBasePairing
IntheWatsonCrickmodel,
thebasepairslieinthesame
plane,orclosetoit.
Tiltofbasenormaltohelical 10.8
axis=1.2
Averagepropellertwist=+16

11.1

What is the distance between

base pairs in DNA?

StackedDimerStackingEnergy(kJ/mol)

StackingEnergiesforthe
tenpossibledimersin
BDNA

C-G
G-C

-61.0

C-G
A-T

-44.0

C-G
T-A

-41.0

G-C
C-G

-40.5

G-C
G-C

-34.6

G-C
A-T

-28.4

T-A
A-T

-27.5

G-C
T-A

-27.5

A-T
A-T

-22.5

A-T
T-A

-16.0

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 chromosome
that carries the muscle protein gene.

Chromosome Painting
Spectral Karyotyping
Each chromosome is hybridized with probes specific to sequences found on that chromosome.

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

This karyotype shows a translocation. The q arm of chromosome 22 has been

attached 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 Analysis

5th ed. table 9-2

Experiment: Chromosome Banding II

1. Before lab, the slides with cells affixed will have been "aged" by
incubation in a 60C 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 (conted)

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 Shellys
Section
Wed. Spring 2010
1000X bright field

Scontras & Schwarz Eric Johnsons section Thurs. 315 Spring 2010
1000X bright field

Susan Shaos section 306 Tues, spring 2009

1000X, bright field

Taras section
Kaitlins lab 3.28.13