Detection of VNTR sequences of human chromosome No.
1 with
polymerase chain reaction
All chromosomes in the human genome contain short tandem repeated sequences.
The copy number of repetition varies in different individuals and is mostly different for
the two homologous chromosomes. It means that a certain chromosomal locus exists in
several forms in a population (i. e. several different alleles are present), depending on the
copy numbers of the repeated sequence. These polymorphic sequences are divided into
two subgroups. Those that contain 2-9 bp long repeating units are called microsatellites1,
the longer 10-60 bp long repeated sequences are the Variable Number Tandem Repeats
(VNTR sequences or minisatellites). Both of them are characteristic examples of human
DNA polymorphism. Detection of a VNTR sequence of a certain chromosomal locus by
polymerase chain reaction (PCR) can serve as a specific genetic marker suitable for
human identification. (The RFLP-based typing systems are also highly informative, but
they require greater than 50 ng of high molecular weight DNA and involve the use of
multiple radioactive probes to obtain a result.)
The D1S80 marker is found on chromosome 1. and consist of repeat units 16 base
pairs (bp) in length. At least 29 alleles have been identified that range from
approximately 350 to 1000 bp and determine as many as 435 possible genotypes. (These
data show the high level of polymorphism characteristic for this locus.) Choosing
appropriate primers hybridizing to flanking nonrepeated sequences two DNA fragments
can be amplified from the two homologous chromosomes No. 1. PCR product size
differences are resolved by employing polyacrylamide gel-electrophoresis and silver
staining and genotypes are interpreted with an allelic ladder (Fig. 1.) In our experiment
we will use agarose gel-electroporesis and ethidium bromide staining, which provide
lower resolution and sensitivity but are easier to perform in the student lab.
The allele frequencies from randomly selected individuals are shown in Table 1.
Alleles 18 and 24 are the most frequent among Caucasians, whereas alleles 21, 22, 28
and 34 occur with higher probability in African Americans. (Allele numbers reflect the
copy number of the repeated sequence.)
Human DNA used in this assay can be obtained from white blood cells, cells of
buccal lining, sperm cells, etc. — apparently any biological material from which DNA
can be isolated. The assay requires about 2.5-10 ng (nanograms) of DNA. Since a
detection method of lower sensitivity is used, we start out from 200 ng of DNA.
The PCR assay can determine whether the tested biological material (e.g. a drop of
blood from the scene of a crime) could originate from a particular person or not. Paternity
also can be established, since a child inherits one of the two alleles of the parent. On the
basis of the allele frequencies given in Table 1. frequency of a certain genotype can be
calculated: e.g. it is 2(0.238)(0.348)2 = 16.6% for the heterozygotes with the most
frequent alleles 18 and 24. In the case of two rare alleles (19 and 32) this value is only
0.013%. It means that one out of six persons has a 18/24 genotype, whereas the frequency
1
Simple Sequence Length Polymorphism (SSLP)
2
2pq = frequency of the heterozygotes in the Hardy-Weinberg equation.
1
of 19/32 is one in 7700. If the (geno)type of the specimen and the tested person matches,
then the tested person is the source of the specimen with a probability of 100-16.6 =
83.4% and 100-0.013 = 99.987%, respectively. The power of discrimination values
calculated for the population groups range from 95 to 98%. If the genotype of the
specimen and the tested person are not matching, he or she can be excluded with a 100%
certainty as the source of the specimen.
Fig. 1. Detection of D1S80 PCR products on a polyacrylamide gel with silver
staining. L: Allelic ladder, shows a band for each allele from 14 to 41; C: control DNA, alleles 18,31; 1:
alleles 24, 37; 2: alleles 18,18; 3: alleles 28, 31; 4: alleles 18, 25 5: alleles 17,28; Allele numbers mean the
copy number of repetition.
Table 1. D1S80 allele frequencies from four population groups
2
The reaction
For our experiment we use human DNA as template, and two, 28 and 29
nucleotide long primers. Let's start the reaction as soon as possible, because it takes 2
to 3 hours to run the program. Add the necessary components to a 0.5 ml microfuge
tube sitting on ice. We have to mix the following components:
_____________________________________________________________________
Volumes to be added Final concentration
_____________________________________________________________________
Sterile distilled water: 57.5 l -
10x PCR buffer* 10 l 1x
dNTP mix** 10 l 200 M
RedTaq DNA polymerase (1U/ l) 2.5 l 2.5 U/100 l
Primer D1S80A 5 l 1 M
Primer D1S80B 5 l 1 M
Human DNA template (20 g/ml) 10 l200 ng/ 100 l
____________________________________________________________________
* 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 11 mM MgCl2, 0.1 % gelatine
** (dATP,dGTP,dCTP,dTTP, 2 mM concentration each)
Pipet very carefully and gently. When all the components were added mix the
reaction by sucking it up 2-3 times into a micropipet, set to 50 l. The components
should be kept on ice all the time.
50 l of mineral oil should be layered on the surface of the reaction mixture in
order to prevent evaporation.
Place the reaction tubes into the thermal cycler programmed with the following
parameters:
1 min 94C (denaturation)
1 min 65C (annealing)
1 min 72C (DNA synthesis)
through 30 cycles
The program lasts about 2.5 hours, therefore it should be started at the
beginning of the lab class. When the reaction is over, the microcentrifuge tubes
should be placed onto -20C and stored until the next week’s class.
3
Electrophoresis
The samples are prepared the following way:
No need to add bromophenol blue to the PCR samples, since with the red
dye in the RedTaq DNA polymerase we can trace the sample during loading and
the gel electrophoresis. We have to add bromophenol blue to the markers.
Sample 1: 20 l of PCR product No. 1.
Sample 2: 15 l of the allelic ladder (2.5-fold dilution)
5 l of bromophenol blue
Sample 3: 20 l of PCR product No. 2.
Sample 4: 15 l of 100 base pair ladder (15-fold dilution)
5 l of bromophenol blue
Load the samples in this order onto a 2% agarose gel and run it in 0.5x TBE
buffer3 with 100-120 Volts until the dye runs at least to the 4/5th of the total
length of the gel. Then place the gel on the UV transilluminator. If the bands are
too faint to be analyzed with the naked eye, take a photograph. Establish the
allelic composition of the tested human DNA samples.
Fig. 2. Detection of D1S80 PCR products on a 2% agarose gel with
ethidiumbromide.
2K, 3 and 10: human DNA samples. 2K: alleles 24, 28, L: allelic ladder, alleles 14, 18, 24,
31, 34, 10: alleles 20,26, , L1: 100 bp ladder
3
0.5x TBE: 0.45 M Tris-borate, 0.001 M EDTA
4
Amplification of DNA from human blood
Appendix to the lab class of 2014
Researchers and clinical laboratories often use DNA isolation kits and PCR kits,
which make easier to perform isolation of DNA and subsequent PCR reactions. In the
lab during the class we will use the Blood cell genomic DNA miniprep kit for DNA
isolation and a PCR kit to carry out the PCR reaction.
DNA extraction
(See the description of EZNA blood cell genomic DNA miniprep kit)
2 extractions per group are scheduled, altogether 4 extractions for both groups.
PCR amplification (steps to be performed)
4 reactions per group are scheduled, altogether 8 reactions for both groups.
Add the following reagents to a thin-walled PCR microcentrifuge tube:
For 1 tube For 2 tubes
1. PCR-mix 10 l 20 l (It is already in the
tube, you have to add the following components.)
2. Sterile distilled water 3 l 6 l
3. Primer 1. (20 M) 1 l 2 l
3. Primer 2. (20 M) 1 l 2 l
5. Human DNA (200 ng) 5 l ___ -___
Altogether: 20 l 30 l
The red PCR-mix contains the 4 nucleoside triphosphates, Taq polymerase and the PCR
buffer. If it did not mix well, gently mix it with pipetting.
If you mixed all the common components of the 2 PCR reactions, transfer 1 x 15 l into
another tubes, leaving 15 l in the original tube.
Add the different human DNA samples to the different tubes, 5 l each.
Add 20 l of mineral oil on the top of the mixture in each tube to prevent evaporation.
Close the tubes firmly and place them into a thermal cycler.
Run thermal cycler with the program “Human”.
94 C 2 min initial denaturation
94 C 45 sec denaturation
65 C 45 sec annealing of primers
72 C 1 min DNA synthesis
through 35-40 cycles
During the next week’s lab we will analyze the PCR products on 2 % agarose gel. It is
enough to load 16 l on the gel, to avoid loading too much mineral oil on the gel.
