Mapping of hereditary diseases by use of PCR 5th semester
Exercise guidelines for LU 509
Mapping of hereditary diseases by use of PCR
Bachelor Degree Programme in Medical Laboratory Technology, Copenhagen. Autumn 2008.
Prepared by Søren Jensen
CONTENTS
Introduction 3
Principles of analysis 4
Mutation analysis 4
Marker analysis 4
Sizes of minisatellites 5
Experimental part 6
Exercise overview 6
Exercise guidelines, part 1 7
Exercise guidelines, part 2 9
Subsequent review and presentation 10
Solutions utilised 10
INTRODUCTION
The purpose of this exercise is to show how to utilise the PCR technique in connection with genetic counselling. This could be relevant in cases where a family has become aware of the presence of a hereditary disease within the family. It would be of interest to the family to identify carriers of the disease among family members and the potential risk of having a child who develops the hereditary disease.
This exercise includes DNA from three families who have requested genetic counselling. Each exercise team shall analyse DNA from one family.
For five members of each family, the exercise includes studying a marker system closely connected to the disease gene of interest and the presence of the most common mutation for this gene. For the other family members results from previously performed marker and mutation analyses are made available.
The exercise is divided into three parts:
· The first part includes setting up marker and mutation analyses. DNA samples from the five family members are made available. The samples are mixed with primers, enzyme etc. and amplified by use of PCR.
· The second part includes visualisation of PCR products from the marker analysis on an agarose gel. The PCR products from the mutation analysis are cut with a restriction enzyme and then visualised on an agarose gel. You are to draw a genealogical tree of ”your” family based on the information available. Once the results are available, they shall be analysed and entered in the genealogical tree along with the other results that are at hand.
· The third part includes subsequent review and presentation. You are to present your family to students from the other exercise teams. A prerequisite for approval of the exercise is that each team has prepared for this subsequent review and presentation to a sufficient degree.
PRINCIPLES OF ANALYSIS
During the course of the exercise two different methods are used to reveal the genotype of each of the family members; mutation analysis and marker analysis. Both analyses are based on the PCR technique.
Mutation analysis
Where the disease gene for a particular hereditary disease is identified, the gene can be analysed for different known mutations.
The best way to do this is to amplify the gene in which the mutation resides.
If a restriction enzyme with a specific cutting site is known and which happens to be located in the same part of the gene as a mutation, this enzyme can be utilised to demonstrate the presence of a particular mutation in the person studied.
The first step is to amplify the sequence around the mutation in question, after which the PCR product is treated with the restriction enzyme. If the mutation is present, an altered enzyme cutting pattern is observed as opposed to what would be the case if the mutation was not present. This is called a mutation analysis, as it includes searching for a particular mutation.
Marker analysis
If the pathogenic mutation in a gene is not known, one can make use of certain polymorphic markers close to the disease gene.
Repeat regions consisting of a variable number of a repeated sequence called VNTR (Variable Number of Tandem Repeats) are found in various locations in the genome. If this sequence is short, i.e. 1-6 base pairs, the region is called a microsatellite. If the sequence is longer, the region is a minisatellite.
If performing a PCR amplification with primers flanking a minisatellite in close proximity to the gene of interest, it will then be possible to distinguish between different alleles, based on the length of the nearby VNTRs. The prerequisite for the use of marker analysis in this case is that the selected VNTR is located so close to the gene of interest that these are inherited together.
Sizes of minisatellites
Two of the families in this exercise are afflicted with the same disease.
In connection with the recessive disease a minisatellite system has been located close to the locus of the pathogenic gene. The minisatellite consists of between 1 and 23 repeat units. Each unit is 12 base pairs long.
No. of repeat units / Size2 / 248 bp
10 / 344 bp
17 / 428 bp
23 / 500 bp
For the remaining family, a study is carried out on another minisatellite system. This system comprises between 4 and 20 repeat units. Each unit is 9 bp long.
No. of repeat units / Size4 / 349 bp
13 / 430 bp
20 / 493 bp
EXPERIMENTAL PART
Exercise overview
Part 1 (approximately 2 hours)
· Setting up PCR marker and mutation analyses.
Part 2 (approximately 4 hours)
· Gel application of the PCR product and start of the electrophoresis. Cutting of the product from the mutation PCR.
· Reading of results and entering them in the genealogical tree.
Part 3 (approximately 1 hour)
· Presentation of results.
Exercise guidelines, part 1
After preparing your laboratory station, pick up the things needed to run your PCR. Remember to mark your strips so that you will be able to find them the following day.
Setting up PCR for mutation analysis
Mix PCR for the mutation analysis according to the following table:
Add ml of reagent
Tube no. / 1 / 2 / 3 / 4 / 5 / 6Red water / 2 / 2 / 2 / 2 / 2 / 3
Polymerase with master mix / 5 / 5 / 5 / 5 / 5 / 5
Primer mix / 2 / 2 / 2 / 2 / 2 / 2
DNA / 1 / 1 / 1 / 1 / 1 / -
· Make sure the various ingredients are mixed thoroughly. Cap the tubes and place them in the PCR machine.
The amplification takes place using the following programme:
1 x / Temp. in oC / TimeInit. denaturation / 95 / 10 min.
35 cycles
Denaturation / 95 / 30 sec.
Annealing / 60 / 30 sec.
Extension / 72 / 1 min.
1 x
Final extension / 72 / 5 min.
Subsequent cooling / 4
Setting up PCR for marker analysis
· Mix PCR according to the following table:
Add ml of reagent
Tube no. / 1 / 2 / 3 / 4 / 5 / 6Red water / 4 / 4 / 4 / 4 / 4 / 6
Polymerase with master mix / 10 / 10 / 10 / 10 / 10 / 10
Primer mix / 4 / 4 / 4 / 4 / 4 / 4
DNA / 2 / 2 / 2 / 2 / 2 / -
· Make sure the various ingredients are mixed thoroughly. Cap the tubes and place them in the PCR machine.
The amplification takes place using the following programme:
1 x / Temp. in oC / TimeInit. denaturation / 95 / 10 min.
35 cycles
Denaturation / 95 / 30 sec.
Annealing / 55 / 30 sec.
Extension / 72 / 1 min.
1 x
Final extension / 72 / 5 min.
Subsequent cooling / 4
Exercise guidelines, part 2
Mutation analysis
1) Prepare the restriction enzyme for cutting by mixing a cutting mix consisting of 6 ml of restriction enzyme, 14 ml of buffer and 50 ml of sterile water.
2) Add 10 ml of cutting mix to each of the six tubes with mutation product.
3) Place the tubes on a heating block preheated to 37oC for one hour.
Marker analysis
Setting up gel electrophoresis.
1) Apply the entire volume of the PCR samples (approximately 20 ml) to the middle row of the agarose gel. Then add 10ml of a 50 bp DNA size marker next to the applied samples.
2) Place the lid on the electrophoresis chamber and then connect the wiring to the power supply. Run the electrophoresis at 100 Volts for approximately 1 hour.
3) Visualise the results using UV light.
Mutation analysis
1) After the restriction cut has been in incubation for 60 minutes, apply the products to a gel.
2) Pipette 20 ml of the six PCR samples into the gel. Pipette 10 ml of size marker next to the products.
3) Run the gel for approximately 60 minutes at 100 Volts.
4) Visualise the results using UV light.
5) Discard the gel in a yellow garbage bin and clean the gel tray. Please leave the analysis site in the same condition as when you arrived.
Part 3
Subsequent review and presentation
During this part of the exercise you are to present your family to the other teams.
The following must be included in your presentation:
1) The genealogical tree of the family with the genotype for each family member.
2) Which type of heredity is seen here?
3) Will a mutation analysis alone be sufficiently informative for your family? Can the mutation analysis be utilised to determine whether the pathogenic mutation has been inherited by the children/the unborn child?
4) Will a marker analysis alone be sufficiently informative for your family? Can this marker be utilised to determine whether the pathogenic mutation has been inherited by the children/the unborn child?
Solutions utilised
Tempase Hot Start Master Mix
Contains:
DNA polymerase, 250 U (0.05 U/ml)
Buffer, 30 mM Tris/HCl, pH 8.05, 100mM KCl
dNTP, 400 mM of each
MgCl2, 5 mM
Gel with ethidium bromide (EtBr)
The gels contain ethidium bromide.
NB! This substance is mutagenic and potentially carcinogenic. Wear gloves at all times!
Must be collected in a special container.
TBE buffer 0.5 x
44 mM Tris/base
44 mM boric acid
20 ml 0.5 M EDTA
Water q.s. to 1 litre
Red water
60 % sucrose (w/v)
0.04 % Cresol Red
Buffers for restriction enzyme cutting
Buffer 1
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
1 mM dithiothreitol, pH 7.9 (25oC)
Buffer 2
1 M KOAc
250 mM Tris-acetate, pH 7.6
100 mM MgOAc
5 mM β-mercaptoethanol
100 mg/ml BSA
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