You are here Home page > Results > Documents in english > NCL - PCR > Procedure of test NCL by PCR

Procedure of test NCL by PCR

16 Votes

NCL is the abbreviation of "Neuronal Ceroid Lipofuscinosis", a metabolic disorder that causes an increasing decease of nerve cells. There are distinct subtypes that differ primarily in two points: firstly, the age of the child, when the first symptoms arise, secondly the celerity of the disease´s proceeding. All forms share the feature, that they are incurable at present.

One out of 25.000-40.000 newborns is struck by the juvenile NCL (jNCL). The disease is caused by an error in the genetic material, which is inherited by an autosomal recessive manner. The affected gene lies on chromosome 16. The error leads to an accumulation of abnormal, adipic like substances in the nerve cells, because the essential structural protein needed for natural metabolism is missing. Thus the cell cannot be cleansed of the harmful substances, arisen from the daily energy production, the neuronal cell gets dirty and dies off. First of all the eyes are concerned, short time later also the brain.

The children lose successively the ability to see, to walk and to act. At the final stage the life-supporting functions cannot be longer sustained.

From http://www.ncl-stiftung.de/englisch/home/index.php

See here an interview with the Benson Family, whose daughter has been affected by the disease :


Here, you have the files of procedure of test for the presence of the genetic mutation at the origin of the disease in pdf and in word.

PDF - 27.7 kb
Procedure of test for the presence of the genetic mutation at the origin of NCL
Word - 56 kb
Procedure of test for the presence of the genetic mutation at the origin of NCL
PDF - 60.5 kb
NCL: Detection of PCR Products by Agarose-Gelelektrophoresis
Word - 937.5 kb
NCL: Detection of PCR Products by Agarose-Gelelektrophoresis

Part 1 (150`)

DNA samples used for this experiment will be usually produced by extraction of different kinds of tissues followed by purification using adsorption column chromatography.

Because of lack of time we obstain from extraction and purificaton of DNA to get pure DNA our experiment.

For our experiment we use six ready prepared DNA samples which are taken from one family: father (F), mother (M), 4 children (C1-C4).
The DNA is well purified and may be used immediately for PCR.

2 working groups (a = pair and b = impair) form one group which has to test the DNA samples of one family.
- Impair group: father and C1, C2
- Pair group: mother and C3, C4

Part 2

Preparing of PCR tubes (30 `)

2.1 Put the 6 PCR tubes into the die adapter tubes and label them as following: F, M, C1, C2, C3, C4
Put the adapter-tubes containing the PCR tubes on ice for cooling.

2.2 Pipet 20 μl of DNA solution of each sample 1-6 into the cooled PCR tubes and pipet the PCR-preparations according to the following diagramm:

Nr Sample DNA Mastermix Primer
1 F 20 μl 25 μl 5 μl
2 M 20 μl 25 μl 5 μl
3 C1 20 μl 25 μl 5 μl
4 C2 20 μl 25 μl 5 μl
5 C3 20 μl 25 μl 5 μl
6 C4 20 μl 25 μl 5 μl

2.4 Mix your PCR-preparations by pipeting up and down or by snipping the tube using your finger.

2.5 Cool your PCR-preparations on ice until starting PCR for amplification! (maximum 20`)

Part 3

Procedure of PCR (3 h)

Carry out the PCR according to the temperature program of the thermocycler!

Pay attention on the position of your samples in the thermocycler to avoid confusion! Make sure that your tubes are correctly closed!

Part 4

Evidence of PCR-products by Agarose-gelelectrophoresis ( 2 h)

4.1 According to your assignment give evidence of the PCR-products by carrying out agarosegelelectrophoresis!
4.2. According to your assignment dye your gels and analyse them!