Banding techniques Flashcards
The best method to process slides for G-banding on the same day that they are dropped is:
Warm the last fix to 37oC during harvest.
Prolong the trypsin treatment time.
Incubate the slide in 60oC for 1-3 hrs before banding (+)
Increase the Giemsa concentration.
You are analyzing a clinical case and discover a small extra marker with satellites. What
staining technique would be most useful in determining the chromosomal origin of this
marker?
QFQ
QFQ
GTG
DAPI/DA
NOR(+)
When using a fluorescein labeled probe during the FISH procedure, the appropriate
counterstain used should be:
5-Bromodeoxyuridine
Ethidium bromide
Propidium iodide(+)
DAPI
When doing sequential staining which of the following is used to remove oil from slides?
Ether
Xylene(+)
Acetic acid
Methanol
What is the best banding technique to distinguish between 47,XY,+18 and 47,XYY?
QFQ(+)
GTG
DAPI/DA
RHG
When the identification of paired chromosomes is the primary goal, which of the following banding techniques should be used?
Q-banding
G-banding(+)
Giemsa staining
C-banding
When the addition of nonhomologous material to the q-arm of chromosome 16 is suspected after G-banding, which of the following banding techniques should be used?
C-banding(+)
Q-banding
R-banding
NOR staining
Which chromosome is most different between C and G banding?
1
9(+)
16
Y
What is not a criteria for G-band identification?
band level
location of bands
location of centromere
location of telomere(+)
What do quinacrine and acridine orange dyes stain?
histones
nonhistones
GC rich DNA
AT rich DNA(+)
Positive heteropyknosis
Chromosome regions which have few genes and contain primarily noncoding regions of DNA remain in a constantly more contracted or supercoiled state. This is because these regions do not actively transcribe mRNA. Positive heteropyknosis implies that these regions will stain darker with DNA stains
negative heteropyknosis
DNA with active transcription of genes is less compacted and is said to have negative heteropyknosis. Regions of negative heteropyknosis will stain lightly with DNA stains
Late Replicating DNA
Dark G and Q bands C bands Light R-bands Positive heteropyknosis Heterochromatin AT rich
Early Replicating DNA
Light G and Q bands Dark R bands Negative heteropyknosis Euchromatin GC rich
Chromosome Banding Code
G(type of banding)
T(general technique)
G(stain used)
GTG = G bands by trypsin using Giemsa QFQ = Q bands by fluorescence using Quinacrine RFA = R bands by fluorescence using Acridine Orange RHG = R bands by heat using Giemsa CBG = C bands by Barium hydroxide using Giemsa
G-banding using trypsin
Slides are treated with a trypsin solution. The treatment time is determined by the following factors:
- age of the slide(Slides become more resistant to trypsin the longer they sit at room temperature (3 days - 4 weeks), the longer they are heat treated (2 hours at 90oC or overnight at 60oC) or the longer they are treated with chemicals that denature or crosslink proteins, such as H2O2.)
- concentration of the trypsin solution (Some investigators use 0.01% crystalline purified trypsin diluted in Hanks without Ca++ or Mg+++ ions. These ions bind to the active site of trypsin and inactivate its enzymatic action. Others use Enzar-T trypsin, a 40x concentrate of porcine pancreatic enzymes (0.1cc in 50 cc of Hank’s = 0.02%)
- pH of the trypsin solution (Trypsin is a pancreatic enzyme which works most effectively at a higher pH (8.5). Trypsin becomes inactive at a pH of 6.8. The best G-banding is achieved at a pH of 7.0 - 7.2 where the pH is just barely active. At a higher pH it becomes too strong and can cause fuzzy, distorted or uneven banding. In addition, the difference between underbanding, optimal, or overbanding becomes a matter of seconds, which is too difficult to control or troubleshoot.)
Q-banding by fluorescence using Quinacrine (QFQ)
Q-banding was the first banding technique to be discovered (Caspersson 1970). Q-banding is induced by staining chromosomes with either Quinacrine dihydrochloride or Quinacrine mustard. Both are fluorescent dyes. Slides are then rinsed and mounted in McIlvaine’s buffer (pH of 5.5 - 6.0). A thin layer of buffer is necessary between the slide and coverslip to 1) swell the chromosomes, 2) increase the contrast between the bands and 3) allow for the travel of fluorescent light to the objective.
QFQ use
Quinacrine preferentially binds to AT rich regions of chromosomes.
Q-banding is very useful in the study of chromosomal variable regions. These regions include the Y chromosome, centromeres of chromosomes 3 and 4 and the satellites of D and G group chromosomes.
Advantage: Extremely quick technique, can finish report the same day you harvest
Disadvantage: Poorer quality of photography, especially true if journal presentation is desired. In addition, the fluorescence is not permanent and fluorescent microscopes are expensive.
Conventional staining
The staining of slides prepared from a cytogenetic harvesting procedure with Giemsa without banding is also useful in the study of human chromosomes. Giemsa is a metachromatic stain. In other words, molecules will stack upon each other. Therefore, darkness of stain is a function of time and concentration. Slides stained for 5 minutes in a 4% Giemsa stain diluted in 0.01 phosphate buffer will give a good solid stain. Banding of chromosomes can distort the morphology of metaphase chromosomes and make it difficult to discern important features such as: primary constrictions (centromeres) secondary constrictions (NORs) fragile sites double minutes satellites (D and G group chromosomes) acquired abnormalities such as dicentrics, rings, fragments, breaks and gaps.
C-banding by Ba(OH2) (CBG)
This technique stains the constitutive heterochromatin which is located around the centromere of all human chromosomes and the constitutive heterochromatin located in the distal long arm of the Y chromosome. Chromosomes 1, 9, 16 and Y generally have the largest C-band regions. These chromosome regions are considered to be genetically inert and contain no functional genes. C-bands are also highly polymorphic.
There are two types of heterochromatin:
Constitutive - contains few to none mendelian genes and is rarely transcribed.
Facultative - euchromatic DNA rendered inactive by cellular processes. (e.g. inactive X in females required for gene dosage compensation).
Technique:
Pretreatment of slides with 0.2N HCL - This acid treatment removes any cytoplasmic debris and some of the histone proteins from the chromosomes.
Extraction of DNA with Ba(OH2) - A saturated Ba(OH2) solution (5% in double deionized water) is a strong alkali that selectively extracts the DNA from the non-C-band regions of DNA.
Incubation in 2xSSC at 60 - 65oC for one hour allows further denaturation and extraction of the DNA in the non-C-band regions.
NOTE: This is a timed reaction. Leaving the slides for too long in the Ba(OH2) solution will result in loss of DNA from the C-band regions as well.
CBG use
C-band as well as Q-band polymorphisms are useful in distinguishing fetal cells from maternal cells, donor cells from host cells, in the identification of marker chromosomes, and in the identification of pericentric and paracentric inversions. Perhaps because these regions are inert, considerable variation (polymorphisms) in size of the C-bands does not seem to affect the phenotype.
Basis of Selective Extraction:
Chromatin is a DNA-histone complex
C-band areas have a tighter interaction with the so called nuclear matrix (that which remains after the removal of all chromatin from the interphase nucleus). The nuclear matrix is comprised of nonhistone proteins of (1) inner nuclear membrane (2) nucleolar matrix and (3) intra-nuclear matrix
Presence of more interaction with nonhistone proteins (support structure for chromatin) prevents or slows extraction of DNA. These regions are highly compact in structure.
NOR stain (Nucleolar Organizer Region)
Silver staining, sometimes called AgNOR staining, stains the NOR's of metaphase chromosomes and the nucleolus of interphase cells. NOR's - nucleolar organizing regions are the chromosomal regions located on chromosomes nos. 13, 14, 15, 21, and 22 in the stalk region. This is the location of the ribosomal cistrons (gene clusters). These ribosomal cistrons code for the 18s and 28s subunits of the ribosomes and are responsible for the formation of the nucleolus in interphase cells. Ribosomes transcribe RNA transcripts into proteins, and the nucleolus is where ribosomal proteins are transcribed. Silver nitrate (AgN03) stains the NOR specific proteins and formic acid or formalin is the catalyst which drives the reaction. Only those NOR's (or cells) which were active in the previous cell cycle will stain positively. These chromosomal regions will contain residual proteins, which will precipitate silver.
Distamycin A - DAPI stain by fluorescence
This is a banding technique that stains only a specific subset of C-bands.
Distamycin A - DAPI stain by fluorescence use
DAPI is a fluorescent dye and Distamycin A is a nonfluorescent antibiotic which binds to AT-rich DNA. Distamycin A quenches all DAPI fluorescence except for the C-band regions of chromosomes 1, 9 16, 15p11 and Yq12. This technique is especially useful in the determination of chromosomal markers originating from the short arm of chromosome 15 and the Y heterochromatin.
Fluorescent in situ hybridization (FISH)
This technique involves hybridization of DNA specific probes on to interphase and metaphase chromosomal DNA.
Satellite probes
Hybridize to specific areas of repetitive DNA (e.g., centromere probes; Y specific probe and short arms of acrocentric chromosomes)
Painting probes
Hybridize to large areas of specific DNA (whole chromosome probes, partial chromosome probes, total genomic)
Unique Sequence probes
Hybridize to small areas containing a specific gene or gene cluster (cosmid probes and telomere specific).
1 Specimen preparation FISH
Painting procedures and mapping of DNA probes on metaphase chromosomes and interphase cells can be performed on conventional methanol/acetic acid fixed cell preparations.
2FISH Denaturation procedures
cause double-stranded DNA to become single-stranded DNA. Single-stranded DNA can hybridize with other single-stranded DNA.
Denaturation of specimen DNA by treatment in 70% Formamide/2x SSC at 70oC (Formamide is an organic solvent that lowers the melting point of DNA which is usually between 90 and 100oC.
Denaturation of the probe.
3FISH Hybridization:
The DNA probe is hybridized onto specimen slides at 37oC for 30 minutes to 16 hours depending on the type of probe used.
4FISH Post Hybridization Wash
This solution is usually 2xSSC or another buffered salt wash and is used to remove free probe or probe bound to non-target DNA. The stringency of the wash solution, or the extent of removal of unwanted probe, minimizes the amount of background, which will be observed under the fluorescent microscope.
NOTE: increasing the temperature or decreasing the salt concentration increases stringency.
5FISH Detection
Direct labeled probes are prelabeled with fluorochromes so this step is unnecessary.
Indirect labeled probes are prelabeled with either the biotin or digoxigenin hapten. These hapten labeled probes can then be detected with avidin or antidigoxigenin antibodies with conjugated fluorochromes. Detection of the probe with antigen-antibody detection systems allows amplification of the signal.
6FISH Probe labeling
Probes are labeled either directly or indirectly with the following fluorochromes:
Fluorescein isothiocyanate (FITC, green) Rhodamine (red) Texas red (deep red)
7FISH Counterstain
A counterstain is used to visualize either the interphase nucleus or the metaphase chromosomes. If the probe is labeled with a red fluorochrome, DAPI is a preferred counterstain (blue). On the other hand, if the probe is labeled with FITC, which produces a yellow-green signal, propidium iodide (PI) is the preferred counterstain (red).
