A karyo type (Greek karyon = kernel, seed or nucleus) is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. OR complete set of chromosomes in a species, or an individual organism.

part of cytogenetics.

1. Length,

2. the position of the centromeres,

3. banding pattern,

4. any differences between the sex chromosomes,

5. any other physical characteristics.

KARYOTYPING

karyogram or idiogram

The study of karyotypes is important for

•  cell biology 

• genetics,

• Clinical genetics

• evolutionary biology 

To study chromosomal

aberrations, cellular function, taxonomic relationships,

and to gather information about past evolutionary events.

• The sex of an unborn fetus can be determined

1. position of centromere - arm length ratio2. secondary constrictions (nucleolar organisers)

1. Short arm is labeled P (French for petit)2. Long arm is labeled Q

Human chromosomes are divided into 7 groups & sex chromosomes

A 1-3 Large metacentric 1,2 or submetacentric

B 4,5 Large submetacentric, all similar

C 6-12, X Medium sized, submetacentric - difficult

D 13-15 medium-sized acrocentric plus satellites

E 16-18 short metacentric 16 or submetacentric 17,18

F 19-20 Short metacentrics

G 21,22,Y Short acrocentrics with satellites. Y no satellites.

A B

STAININGThe study of karyotypes is made possible by 

1.  cells (WBCs) arrested during cell division by a solution of colchicine.2. Staining, Usually, a suitable dye, such as Giemsa

Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules. Availability of tubulin is essential to mitosis, and therefore colchicine effectively functions as a "mitotic poison" or spindle poison.

For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Sometimes observations may be made on non-dividing (interphase) cells.

Observations

6 different characteristics of karyotypes are usually observed and

compared

1. Differences in absolute sizes of chromosomes. 

Chromosomes can vary in absolute size by as much as twenty-

fold between genera of the same family.

2. Differences in the position of centromeres.  These

differences probably came about through translocations.

3. Differences in relative size of chromosomes.  These

differences probably arose from segmental interchange of

unequal lengths.

4. Differences in basic number of chromosomes.

these differences could have resulted from successive unequal translocations

which removed all the essential genetic material from a chromosome,

permitting its loss without penalty to the organism (the dislocation hypothesis)

or through fusion.  Humans have one pair fewer chromosomes than the great

apes.  Human chromosome 2 appears to have resulted from the fusion of two

ancestral chromosomes, and many of the genes of those two original

chromosomes have been translocated to other chromosomes.

5. Differences in number and position of satellites.

Satellites are small bodies attached to a chromosome by a thin thread

6. Differences in degree and distribution of heterochromatic regions. 

Heterochromatin stains darker than euchromatin.  Heterochromatin is

packed tighter.  Heterochromatin consists mainly of genetically inactive and

repetitive DNA sequences.  Euchromatin is usually under active

transcription.

Fundamental number

The fundamental number, FN, of a karyotype is the number of visible major

chromosomal arms per set of chromosomes. Thus, FN ≤ 2 x 2n, why???

Humans have FN ???

(acrocentric chromosome pairs: 13, 14, 15, 21 and 22)

CHR NO Changes during development

Instead of the usual gene repression, some organisms go in for

large-scale elimination of heterochromatin, or other kinds of

visible adjustment to the karyotype.

•Chromosome elimination. In some species, as in many sciarid

flies, entire chromosomes are eliminated during development.

•Chromatin diminution . In this process, found in

some roundworms, portions of the chromosomes are cast away

in particular cells. This process is a carefully organised genome

rearrangement where new telomeres are constructed and certain

heterochromatin regions are lost. In A. suum, all the somatic cell

precursors undergo chromatin diminution.

•X-inactivation. The inactivation of one X chromosome takes place

during the early development of mammals

In placental mammals, the inactivation is random as between the two

Xs; thus the mammalian female is a mosaic in respect of her X

chromosomes. In marsupials it is always the paternal X which is

inactivated. In human females some 15% of somatic cells escape

inactivation.

Ploidy: number of complete sets of chromosomes in a cell.

•Polyploidy, more than two sets of homologous chromosomes in the cells,

occurs mainly in plants. major significance in plant evolution  

•Haplo-diploidy: where one sex is diploid, and the other haploid. in

the Hymenoptera.

•Endopolyploidy occurs when in adult differentiated tissues the cells have

ceased to divide by mitosis, but the nuclei contain more than the

original somatic number of chromosomes. e.g the

endocycle (endomitosis or endoreduplication) chromosomes in a 'resting'

nucleus undergo reduplication, the daughter chromosomes separating from

each other inside an intact nuclear membrane.

Aneuploidy:Aneuploidy is the condition in which the chromosome number in

the cells is not the typical number for the species

G-banding is obtained with Giemsa stain following digestion of

chromosomes with trypsin. It yields a series of lightly and darkly stained

bands —

the dark regions tend to be heterochromatic, late-replicating and AT rich.

The light regions tend to be euchromatic, early-replicating and GC rich.

This method will normally produce 300–400 bands in a normal, human

genome.

R-banding is the reverse of G-banding (the R stands for "reverse"). The

dark regions are euchromatic (GC rich regions) and the bright regions

are heterochromatic (AT rich regions).

classic" karyotype, CHROMOSOMAL STAINING

•C-banding: stains centromeres.

•Q-banding is a fluorescent pattern obtained using quinacrine for

staining. The pattern of bands is very similar to that seen in G-

banding.

•T-banding: visualize telomeres.

•Silver staining: Silver nitrate stains the nucleolar organization

region-associated protein. This yields a dark region where the

silver is deposited, denoting the activity of rRNA genes within the

NOR.

molecular cytogenetic technique used to simultaneously visualize all the

pairs of chromosomes in an organism in different colors. 

•Fluorescently labeled probes

•Mixtures

SPECTRAL KARYOTYPING

Digital karyotyping is a technique used to quantify the DNA copy

number on a genomic scale. Short sequences of DNA from specific

loci all over the genome are isolated and typed.

DIGITAL KARYOTYPING/ VIRTUAL KARYOTYPING

A restriction map is a map of known restriction sites within a

sequence of DNA

RESTRICTION MAPPING

Restriction mapping steps

1. Breaking DNA into pieces

2. identifying the locations of the breakpoints.

the process of obtaining structural information on a piece of

DNA by the use of restriction enzymes.

Restriction Enzymes

Recognizes Palindromic seq,

each strand of the DNA can self-anneal and the DNA forms a small cruciform

structure

Hundreds of restriction enzymes that have been isolated and each one

recognizes its own specific nucleotide sequence.

endonucleases that recognize specific 4 to 8 base regions of DNA. restriction sites.

evolved as a bacterial defense against DNA bacteriophage

Sites for each restriction enzyme are distributed randomly throughout a

particular DNA stretch. Digestion of DNA by restriction enzymes is very

reproducible; every time a specific piece of DNA is cut by a specific enzyme,

the same pattern of digestion will occur.

Uses of Restriction Mappingfor many techniques used to manipulate DNA.

One application is to cut a large piece of DNA into smaller fragments to allow it to be

sequenced. Genes and cDNAs can be thousands of kilobases long (megabases -

Mb); however, they can only be sequenced 400 bases at a time. DNA must be

chopped up into smaller pieces and sub cloned to perform the sequencing.

Also, restriction mapping is an easy way to compare DNA fragments without

having any information of their nucleotide sequence.

The sum of the individual fragments =size of the original fragment

If not there are two likely problems.

In one case, some of the smaller fragments may have run off the

end of the gel.

1. the gel was not dense enough and therefore was unable to

resolve fragments close in size.

2. This leads to a lack of separation of fragments which were close

in size.