Chromatin refers to "colored" material in the nucleus. We can think of it as chromosomal material. Heterochromatin refers to non transcriptionally active chromatin. It can be facultative heterochromatin such as the X chromosome which is capable of being active transcriptionally although it is not always active or constitutive heterochromatin such as the q arm of the Y chromosome or the centromeric heterochromatin which is never transcriptionally active. Euchromatin refers to transcriptionally active chromatin.

Karyotyping is the process of preparing chromosomes for analysis. The tissues used vary but the most common tissues sampled are lymphocytes (white blood cells), skin fibroblasts, amniotic fluid cells, gonadal tissue, chorionic villi (CVS) and bone marrow. Direct preparations without the use of a mitogen are made on bone marrow, lymphocytes, gonadal tissue, CVS, and tumors. When cultured, cells are placed in a nutrient solution containing amino acids and other nutrients, a pH indicator, antibiotics, with or without mitogens (PHA or pokeweed mitogen) and then grown at 37^°C in CO₂ atmosphere. Lymphocytes are cultured three days and amniotic cells, fibroblasts, CVS, and other fetal tissues are cultured ten days. Colcemid, which inhibits the assembly of microtubules of the spindle, is added prior to harvest to stop mitosis and to collect sufficient cells with prophase-prometaphase chromosomes. Cells may be grown on microscope slides in sterile chambers or they may be attached to the flask in which they are grown. If release of cells from substrate is necessary, trypsin and EDTA are added. The cells are then centrifuged to remove the culture medium and/or the trypsin and EDTA. A hypotonic solution is added to swell the cells followed by centrifugation and fixation in methanol:acetic acid. The cells are dropped onto slides if they were not grown on slides. Most banding procedures require pretreatment before staining. Trypsin and Giemsa produce G-banded chromosomes (GTG).

The slides are scanned for metaphase spreads and usually 10 to 30 cells are analyzed under the microscope by a cytogeneticist. When a good spread (minimum number of overlapping chromosomes) is found a photograph is taken or the analysis is done by a computer. The chromosomes are arranged in a standard presentation format of longest to shortest. Actually chromosome 21 is smaller than chromosome 22. In a standard karyotype, the chromosomes are arranged by size and location of the centromere. There are 7 groups: A, 1 - 3 long metacentrics; B, 4 - 5 long sub-metacentrics; C, 6 - 12 sub-metacentrics; D, 13 - 15 acrocentrics which may have satellites (normal chromosome polymorphisms) on the p arm; E, 16 - 18, sub-metacentrics; F, 19 - 20, short metacentrics; G, 21 - 22, acrocentrics with or without satellites (normal polymorphism) on the p arm; the X chromosome is a sub-metacentric which belongs in the C group and the Y chromosome is a sub-metacentric which can be placed in the G group on the basis of size. The shorter arm is placed on top in a karyotype and is called the p arm, the longer arm is on the bottom and is called the q arm.

There is a standardized way of denoting the bands on banded chromosomes. The karyotypes of normal individuals are almost identical with the exception of some chromosome polymorphisms such as the satellite of the D and G group chromosomes and some heterochromatic regions around the centromeres of 1, 9, 16 and Yq.

The chromosomes of the great apes are remarkably similar to ours except for some chromosome rearrangements. Chimps, orangutans and gorillas have 48 instead of 46. The human chromosome #2 is the result of a translocation of two chromosomes found in the great apes. There has been a Down Syndrome chimpanzee with an extra small chromosome homologous to our 21.

Banding techniques: G (pretreatment with trypsin then Giemsa stain); Q (quinicrine, fluorescent bands); R (reverse banding) requires heating in a saline buffer prior to staining with Giemsa or Quinicrine and is often called T (telomeric) banding since it also highlights the telomeres; C (Centromere or Constitutive heterochromatin) stains the heterochromatin in the centromeres, especially of chromosomes 1, 9, and 16; NOR (nucleolar organizing regions) silver nitrate stains selectively the satellite stalks of the acrocentric chromosomes; SCE (sister chromatid exchanges) requires replication in 5 BudR, an analog of thymidine followed by Höescht stain.

The major dark and light areas starting from the centromere have numbers. Each band is numbered beginning with 11 or 11.1 at the centromere. When you see numbers assigned, the first number is the region, the second is the band within the region, and the third number is a sub-band within the band. For example: 31.2 means 3 is the region, 1 is the band, 0.2 is the sub-band. Regular banding produces about 550 light and dark bands. High resolution banding (HRB) to look for microdeletions requires the cell culture to be partially synchronized to catch the cells in pro-metaphase before they condense too much. It produces about 800 ⁺ bands

Dark G bands and bright Q bands are AT rich, late replicating, gene poor, contain LINE repeats. G light bands and dull Q bands are GC rich, early replicating, gene rich, contain SINE/Alu sequences

Each chromosome arm is numbered beginning with the centromere: 11 or 11.1 at the centromere, e.g., 21.2 : Region = 2, Band = 1 and sub band = 0.2

46, XX is the normal female karyotype; 46, XY is the normal male karyotype. X and Y are the sex chromosomes; all others are called autosomes. 47,XY,+21 means there is an extra #21 chromosome (a trisomy); 45,X, means there is one less chromosome, namely one sex chromosome is missing (a monosomy). Mosaicism is denoted by a "/", e.g., 45,X/46,XX indicates a Turner female with two cell lines, one normal and one missing the second X. This arises from post zygotic non disjunction, after fertilization and in mitosis of the zygote or embryo.

Haploid = n is the number of pairs of chromosomes. Most organisms are diploid, 2n, and their gametes are haploid, n. Polyploidy refers to having multiple sets of chromosomes: 3n (triploid); 4n (tetraploid). Aneuploidy refers to having (usually) one more or less of the diploid number of chromosomes: trisomies (one extra); monosomies (one less).

FISH (fluorescence in situ hybridization) has become a commonly used staining technique where known sequences of DNA are fluorescently labeled and hybridized to chromosomes.The process depends on complementary base pairing, similar to Southern blots. In situ refers to the fact that the fluorescent DNA probe is hybridized to the DNA of interphase nuclei or metaphase chromosomes which have been fixed on a slide. FISH uses locus specific DNA probes, repetitive DNA such as the centromeric alpha satellite DNA or whole chromosome painting (spectral painting). It can be used in the identification of marker chromosomes, rings and other structural anomalies using probes which are specific for each chromosome (painting probes). You saw an overhead of a 3p+ where the "+" was shown to come from chromosome 13; an iso 5q which was actually a 5 with a piece of 6; an 8 p+ where the + was a piece of 21; an 8 p+ where the + was a piece of 22. FISH is now used almost routinely for microdeletion syndrome which we will discuss later.

Centromeric repeat probes which bind to the unique satellite sequences give a large, diffuse but intense signal around the centromere. They can detect aneuploidies such as trisomies 21, 18, 13 even in interphase nuclei. They are not useful in detecting structural abnormalities.

Locus specific probes give a sharp, discrete signal at the relevant positions in each chromatid of a metaphase chromosome; they can be used to identify or confirm microdeletion (or macrodeletion) syndromes.

Painting probes uniformly decorate the entire chromosome or can be designed to paint the p or q arm, etc. The DNA sequences used are unique to the chromosome or arm painted. They can be used to identify the chromosomal origin in structural rearrangements such as translocations, rings, isochromosomes, etc. The term, "spectral karyotyping" is used to describe "whole" chromosome specific painting.