Materials Included In Kit

Additional Materials Required

Prelab Preparation

Copy one Karyotype sheet, one Denver System Worksheet and the student portion of the activity for each student group.

Safety Precautions

Although the materials are not considered hazardous, please follow all laboratory safety guidelines. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

Disposal

The pieces of paper may be disposed of in the normal trash.

Lab Hints

• Enough materials are provided in this kit for 30 students working in groups of three or for 10 groups of students. All materials are reusable. This laboratory activity can reasonably be completed in one 50-minute class period. A small group size or a larger number of groups may be accommodated by making multiple copies of each Karyotyping Sheet for use by more than one group.
• Groups will complete the activity at different times; extend the activity into a research project by assigning specific genetic disorders or techniques to students for further inquiry.
• Copy the sample ideograms on page 8 to an overhead for easy reference by the students.
• To create a karyotype, actively dividing cells are treated with a chemical that interrupts the microtubule network responsible for chromosome movement during anaphase. In the absence of microtubules, the chromosomes remain near the equator of the cell and cell division is halted in metaphase. The metaphase cells are then exposed to a hypotonic (low salt) solution to swell the nuclear membranes—this causes the chromosomes to spread apart within the nucleus. Next, the chromosomes are stained with one of several specialized stains. These bind to specific areas in the highly condensed chromosomes, creating light and dark bands that resemble a bar code pattern. The stained cells are viewed at 1000X with a light microscope and photographs are taken of 15 to 20 representative cells. Each photograph is then cut and arranged on a Denver System worksheet. The resulting karyotype is analyzed to determine if the organism has a normal genetic composition or if any irregularities or problems exist.

Teacher Tips

• Chromosome 21 actually contains fewer base pairs, and is therefore shorter, than chromosome 22. This was not known in 1961 when the Denver System was established.
• G-Banding with Giemsa stain is the most common staining or banding procedure used by geneticists. A newer technique called Fluorescent in situ hybridization (FISH) is becoming very popular as well.
• Call Flinn Scientific to obtain a complimentary copy of the Human Genome Poster distributed by the U.S. Department of Energy, Human Genome Program.

Answers to Prelab Questions

1. In human chromosomes, the centromere is located in one of three general locations. Draw and label three different chromosomes to show the three possible centromere positions.
   {10811_PreLabAnswers_Figure_1}
2. Describe the two major genetic abnormalities that may be observed through karyotyping.

   Numerical errors include trisomy, in which three copies of a chromosome are present instead of the usual two, and monosomy, in which only one copy of a chromosome is present. Structural errors occur when part of a chromosome is missing or moved from its correct position.

3. What is the genetic composition of a normal male?

   46,XY

Sample Data

Karyotype 1
Disomy Y syndrome Male—47,XYY—two copies of 1–22, one copy of X, two copies of Y
Karyotype 2
Down syndrome Male—47,XY,21—two copies of 1–20 plus 22, one copy of X, one copy Y, three copies of 21
Karyotype 3
Edwards syndrome Female—47,XX,18—two copies of 1–17 plus 19–22, two copies of X, three copies of 18
Karyotype 4
Klinefelter syndrome Male—47,XXY—two copies of 1–22, two copies of X, one copy Y
Karyotype 5
Normal Female—46,XX—two copies of 1–22, two copies of X
Karyotype 6
Normal Male—46, XY—two copies of 1–22, one copy of X, one copy Y
Karyotype 7
Patau syndrome Male—47,XY,13—two copies of 1–12 plus 14–22, one copy of X, one copy Y, three copies of 13
Karyotype 8
Robertson Female—45,XX,21—two copies of 1–20 plus 22, two copies of X, the two 21s stuck together at their p arms (see Figure 2)
Karyotype 9
Triple X syndrome Female 47,XXX—two copies of 1–22, three copies of X
Karyotype 10
Turner syndrome Female—45,XO—two copies of 1–22, one copy of X Sample ideograms for each chromosome

Teacher Handouts

10811_Teacher1.pdf

References

http://rarediseases.info.nih.gov/default.htm (accessed December 2006)

http://ghr.nlm.nih.gov/ (accessed December 2006)

http://medlineplus.gov/ (accessed December 2006)

http://www.medgen.ubc.ca/wrobinson/mosaic/index.htm (accessed December 2006)

Introduction

When a cell undergoes mitosis its nuclear DNA is tightly coiled into structures called chromosomes. Geneticists can inspect the chromosomes for genetic abnormalities by staining the chromosomes and viewing them using an oil immersion lens on a compound microscope. This technique, called karyotyping, allows geneticists to see abnormalities, such as extra chromosomes, missing chromosomes and malformed chromosomes.

Concepts

• Centromeres
• DNA banding
• Chromosomal abnormalities
• Karyotyping

Background

Normal human somatic (body) cells have 46 chromosomes. The 46 chromosomes include two possible types of sex chromosomes, X and Y, and 22 pairs of autosomes. One copy of each autosome and an X chromosome are inherited from the mother. The father provides a second copy of each autosome plus either an X or Y sex chromosome. If the resulting embryo has two X chromosomes it is a female, while an embryo with both an X and Y sex chromosome will become a male. The autosomes pair up in so-called homologous or matching chromosomes. After staining, each pair of homologous chromosomes is easily distinguished from other chromosomes by differences in length, in the position of the centromere, and by the pattern of bands created using special stains. The centromere is always located in one of three possible positions in human chromosomes (see Figure 1). If the centromere is in the center of the chromosome it is called metacentric. If the centromere is located near one end of the chromosome it is called acrocentric. The third centromere position may be between the center and the end of the chromosome—this position is called submetacentric.

In order to facilitate comparison of the genetic makeup of people from all over the world, geneticists established a classification and naming system, called the Denver System, to describe and identify chromosomes. The Denver System was established in 1961 at an international meeting of geneticists in Denver, Colorado. According to the Denver System, the sex chromosomes are named X and Y, while the autosomes are numbered in descending order, with the largest called chromosome 1. and the smallest chromosome 22. The Denver System further subdivides or classifies the chromosomes into eight groups A–G (see Table 2). A karyotype is the specific arrangement of specially stained chromosomes using the Denver System. Problems occurring during mitosis and meiosis can result in cells containing too many or too few chromosomes or parts of chromosomes. Other problems may occur if part of one chromosome breaks off and becomes attached to a different chromosome. The consequences of chromosomal abnormalities can vary from fatal or to insignificant, depending upon the size and location of the error and when the error occurred. For example, an embryo with three copies of chromosome 1 will result in a miscarriage, whereas cancer may occur when specific genes are deleted from chromosomes during mitosis in an adult.

There are two basic types of genetic abnormalities that can be detected by geneticists using a karyotype—numerical errors and structural errors (see Figure 3). Numerical errors include trisomy, in which three copies of a chromosome are present instead of the usual two, and monosomy, in which only one copy of a chromosome is present. Structural errors occur when part of a chromosome is missing or not located in its correct position. There are four types of structural errors—translocations, inversions, deletions, and duplications. Translocations arise when part of one chromosome breaks off and attaches to another chromosome. Chromosomes with acrocentric centromeres (centromere near one end) such as chromosomes 13, 14, 15, 21 and 22 have very short p arms that break off rather easily. The remaining long q arm may translocate and stick to another acrocentric chromosome (see Figure 2). Inversions involve a section of chromosome breaking off and then reattaching to the same chromosome upside down. If a section of a chromosome is completely absent it is called a deletion. Duplications occur when a section of the chromosome is repeated. In some cases the chromosomal abnormality is present in the gamete (egg or sperm cell) due to problems in meiosis. A faulty gamete will produce an embryo in which every cell contains the abnormality. Full numerical abnormalities are usually fatal and the embryo miscarries. The few exceptions are Monosomy X (one X), Disomy Y (two Ys), and Trisomy 13, 18, 21 or X (three copies of the same chromosome). Surviving full structural abnormalities are not always severe because the extent of the change from “normal” varies.

Mosaicism occurs if the chromosomal abnormality occurs during mitosis in the embryo. A mosaic is one organism with two different genotypes—some of the cells will have a normal set of chromosomes, while others will have the abnormal set of chromosomes. The severity of abnormality in a mosaic can be mild or severe depending upon how old the embryo is when the error occurs and how much of the chromosome is in error. If the error occurs late in development, very few cells will carry the error and the organism will have mostly normal-functioning cells with either mild problems or even no problems. If the error occurs early in development then a majority of cells will carry the error, potentially resulting in severe problems such as mental retardation and heart defects.

The karyotypes of a normal male and female, as well as individuals with different numerical errors are provided in this activity. Table 2 summarizes the genetic composition of each individual whose karyotype is included in this activity. The genetic composition of the karyotype is summarized as follows: first, by the number of chromosomes present in the cell (45, 46 or 47); then by the sex chromosomes present (XX, XY); and finally by the number of any abnormal chromosome (e.g., 21, X). For example, 47,XXY is a male with two X chromosomes and one Y chromosome instead of the usual one X and one Y.

Experiment Overview

The purpose of this activity is to analyze a simulated karyotype, called an ideogram, in order to identify the genetic composition of an individual.

Materials

Prelab Questions

1. In human chromosomes, the centromere is located in one of three general locations. Draw and label three different chromosomes to show the three possible centromere positions.
2. Describe the two major genetic abnormalities that may be observed through karyotyping.
3. What is the genetic composition of a normal male?

Safety Precautions

Although the materials are not considered hazardous, please follow all laboratory safety guidelines. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Record the Karyotype Sheet number on the Denver System Worksheet.
2. Count the chromosomes on the Karyotype Sheet to determine the total number of chromosomes and record the results on the Denver System Worksheet.
3. Using scissors, carefully cut out the individual chromosomes on the Karyotype Sheet.
4. Arrange the chromosomes in order of decreasing size, from largest to smallest. (That is how they will be arranged on the Denver System Worksheet.)
5. Use the size, centromere location, and banding pattern on each chromosome to match homologous pairs of chromosomes, and place the matching pairs on the Denver System Worksheet, with the centromere on the line provided and the short p arm above the line. Note: Refer to Table 1 for the centromere locations on human chromosomes.
6. Tape the chromosomes to the Denver System Worksheet. Answer the question on the Denver System Worksheet.

Student Worksheet PDF

10811_Student1.pdf