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Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation
Research Assistant Wanted

Brother Gregory wants you to help him find the answer questions about DNA extracted from various materials and specimens. In these investigations you will be provided with DNA taken from unknown materials. You will be asked to find out the base composition of these DNA molecules and use this information to identify the unknown materials.

First, print out your personal investigation page (below) and find out which specimens and questions you are supposed to investigate and answer. Carry out the experiment, gather and record your data, analyze your results and write down the appropriate numbers on your personal investigation page.

This is important. Bring your completed investigation page to the examination, if it is required on your topic schedule.




Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation
The Problem

Specimens of unknown materials have been taken from an Egyptian mummy. It is hoped that they will give some clues as to what killed the Pharaoh Horemheb.

One clue will be the chemical nature of the DNA molecules found in these specimens. DNA molecules have characteristic amounts of G+C and A+T. This is called the base composition of the DNA, and is usually written in the form "%GC".

For example:
"DNA from specimen X has a base composition of 45%GC".

There are various ways of determining the base composition of DNA molecules. In this investigation you will use
Poly Lysine Kieselguhr (PLK) Chromatography.

After determining the DNA base composition of the unknown specimens, you will then determine the base composition of DNA taken from a variety of organisms. It is possible that you will find a match between the base composition of your unknown specimens and the properly identified, known, organisms.




Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation
The Method

DNA from the known, and unknown, specimens and organisms are tightly bonded to a synthetic polymer of the amino acid lysine (poly-L-lysine).

The polylysine is in turn absorbed onto the surface of a white powder called kieselguhr, making a semi-solid matrix which traps the DNA.

When salt solutions of various strengths are washed though a column of PLK/DNA the bonds between the polylysine and the DNA begin to break down, and the freed DNA is released.

get more details
about PLK
chromatography
The concentration of salt in the solution needed to release the DNA depends on the strength of the bonding between the polylysine and the DNA, which in turn depends on the base composition of the DNA (%GC).

After placing the polylysine/DNA/kieselguhr-complex into a glass tube, stronger and stronger (more concentrated) salt solutions are passed through the column and over the complex. As the DNA molecules are released they are washed out of the bottom of the tube and collected in test tubes (fractions).

The amount of DNA in each tube (fraction) can be measured and an "elution profile" determined. The fraction number at which the maximum amount of DNA is released from the complex also give as measure of the base composition (%GC) of the DNA.


Step One:


Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation
The Standard Curve

As the first step in this investigation, it is necessary to construct a standard curve, which shows the relationship between the fraction number at which standard DNA samples wash off (elute) from a PLK column, and the base composition (%GC) of the DNA molecules.

Procedure

  1. Print out the Standard Curve page on which you will record your results and plot your standard curve graph.

  2. Go to the DNA base composition analysis screen.

  3. At the bottom of the screen, to the right, there is a brown area with a place for you to enter a "%GC" value which is entitled enter standard DNA value. Put any number from 1 - 100 in this box (IMPORTANT NOTE: do NOT add the "%" sign!!) at the bottom of the screen,
    and click ---*** Start ***---.

  4. You will see an "elution profile" in which the red bars represent the amount of DNA that is being washed off the PLK column at that particular fraction number. You will need to find the fraction number at which the peak value (i.e. the most DNA) is being washed off the column.

  5. Carefully note the fraction number at which this maximum amount of standard DNA is being washed off the PLK column. Write it down!

  6. Re-click the "Start" button a few times. Each time, write down the fraction number at which the maximum amount of DNA is being washed off (eluted) from the PLK column.

  7. Using your "standard curve printout" write down and record two values; the value for the base composition of the DNA being tested (the number you put in the box for %GC at the bottom of the screen) and the fraction number at which the DNA was washed off (eluted) from the PLK column.

  8. Change the number representing the %GC, standard DNA value, in the box and repeat the analysis. Once again record both the fraction number and the %GC on your data table.

  9. Continue repeating this procedure until you have a good range of values. For example, 5 - 10 values in the range 30 - 90 %GC would probably be best.

  10. Using this set of values, plot a graph that shows the relationship between the %GC and the fraction number at which the DNA washes off the PLK column. (See small diagram, above left).


Step Two:

Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation

The Specimens

As the second step in this investigation you now use the PLK column to find the base composition (%GC) of the specimens you have been assigned (printout your personal investigation page - below - to find out which are your specimens).

Procedure

  1. First, print out your Personal Investigation Page (see below), and find out what specimens you have been assigned.

  2. Go to the DNA base composition analysis screen.

  3. Click on the appropriate specimen number(s) on the left side of the page. Use the numbers corresponding to your assigned specimens and numbers.

  4. Click on the Start button at the bottom of the page (below the selections). Do this several times.

  5. Record the fraction number at which the maximum amount of DNA is washed off the PLK column.

  6. Use your "Standard Curve" to find the base composition (%GC) of your specimen(s).

  7. Look for the fraction number at which your specimen DNA was eluted from the column. Move directly up to the curve (line) you have plotted on the graph paper. The move to the left and find the %GC for that fraction number. This is the value you want.

  8. Put these values on your Personal Investigation Page (see below) in the appropriate answer boxes.


Step Three:

Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
* Personal Investigation
Known and Identified Organisms

As the third step in this investigation you now use the PLK column to find the base composition (%GC) of the DNA in known organisms that are already identified. You will then compare the base composition of your specimens (from your Personal Investigation Page) with those of known, identified organisms.

From this comparison, you may be able to find out what killed the Pharaoh Horemheb - but you never know!

Procedure

  1. Go to the
    DNA base composition analysis - Known Substances
    screen.

  2. You will see a list of organisms on the left. Click on the one you want to use.

  3. Click on the --***Start***-- button several times and make a note of the fraction number at which most of the DNA washes off (elutes) from the PLK column. Write this down.

  4. Use the Standard Curve graph to determine the base composition (%GC) of the known and identified organisms.

  5. See if any of them match your assigned specimens from your Personal Investigation Page


Print out your
personal investigation page

Page Items
* Research Assistants
* The Problem
* The Method
* The Standard Curve
* The Specimens
* The Knowns
Carefully enter your Seat Number or PCIN number
(e.g.
MM34, or MA56, or WA41)
in the box below, click, and print out the page that appears.

Find the answers to the questions, write your answers on the investigation page, and bring the completed assignment to the exam for grading purposes.


Personal Investigation Page

Enter your seat number of PCIN number here: -


PLK and Unknown Specimens

PLK and Identified Specimens


Science@a Distance
© 2003, Professor John Blamire