P4a-IV: Biochemie - DNA-profile analysis
IV
1
DNA-PROFILE ANALYSIS:
KARAKTERISERING van één MOLECUUL
RECOMMENDED READINGS:
L. Stryer, Biochemistry, Fifth Ed. (2002), Freeman (San Francisco), Chapter 5 and 6.
SYNOPSIS
In this experiment we will isolate chromosomal DNA from scrapings from the inside of the
cheek. Next, a certain fragment (locus) of this DNA will be amplified by a polymerase
chain reaction (PCR). After separation of the newly formed DNA fragments by agarose gel
electrophoresis and staining, we can compare the length of this locus between the different
individuals. The variability in length can form the basis of DNA profile analysis.
I
INTRODUCTION
DNA, in all forms of life, is a polymer made up of a large number of deoxyribonucleotides,
each composed of a base, a sugar, and a phosphate group. The bases of DNA molecules
carry genetic information, whereas their sugar and phosphate groups perform a structural
role. The bases contain four major types of heterocyclic nitrogen compounds, adenine,
thymine, guanine, and cytosine. The nucleotides are held together by 3',5'-phosphodiester
bonds (Fig. DNA / PCR.1). The quantitative ratio and sequence of bases vary with the
source of DNA. Native DNA exists as two complementary strands held together by
hydrogen bonds and arranged in a double helix (Fig. DNA / PCR.2).
The total genetic information carried by a cell or organism is called genome. In eukaryotic
cells the genome is divided into several, or many, chromosomes, each of which contains a
very large, linear DNA molecule. Whereas most prokaryotes are haploid (containing only
one copy of their chromosome), most eukaryotic cells are diploid; that is, they carry two
copies of each chromosome. For example, the human genome is made up of 23 different
chromosome pairs, so that normal, diploid human cells have a total chromosome number
of 46. Of each pair of chromosome one is inherited from the mother, and the other one
from the father.
DNA of the human somatic cell nucleus contains over 3 x 109 base pairs. About 5 % of this
DNA encodes for more than 30,000 genes, while the remainder of the DNA is non-coding.
Until now the function of this non–coding DNA is not known. The coding DNA is more
or less identical for all individuals. The non–coding DNA can differ in base sequences
between individuals.
The genetic information on a locus (a special place on the chromosome) is present in
duplicate, once on each chromosome. Genetic variations of a certain locus are called
alleles. If the alleles on both chromosomes are different, the organism is classified as
heterozygous, when both alleles are identical it is homozygous.
2
P4a-IV: Biochemie - DNA-profile analysis
Fig. DNA / PCR.1. Polynucleotide structure of DNA.
Fig. DNA / PCR.2. Helical structure of DNA.
P4a-IV: Biochemie - DNA-profile analysis
3
DNA profile analysis focuses on variable regions of DNA of individuals. One such form of
variability is called VNTR (variable number of tandem repeats). These VNTRs are
families of repetitive sequences found at different loci which vary not simply as to
sequence but also as to how many repeats of an individual sequence occurred at a single
site on a particular chromosome. The greater the variability in the number of these repeats,
the greater the change to discriminate between individuals on basis of DNA length
patterns.
The use of VNTRs in DNA fingerprinting is illustrated in Fig. DNA / PCR.3.
Fig. DNA / PCR.3. Two methods of DNA profile analysis. (a) Two different alleles of a region of a
single chromosome. The alleles differ only in the number of repeats in the VNTR. DNA from cells
containing these chromosomes can be cut with a restriction enzyme E (which does not cut within the VNTR)
and the fragments are separated on an agarose gel. The fragments containing the VNTR are then identified
after Southern blotting by hybridization with a probe specific for that VNTR. (For simplicity the figure
shows only the result from individuals whose two chromosomes each have the same allele at this site.) (b)
The same alleles, but with primers that could be used to amplify the VNTR segments by PCR. The products
of the PCR reaction can be loaded directly onto the gel without restriction digestion.
In this figure method (a) is also called Restriction Fragment Length Polymorfism
(RLFP) Analysis to detect VNTR alleles. For this method one needs a labelled probe to
detect the fragments on the blot. Moreover one needs more material of the chromosomal
DNA than in method (b), for in the last method the alleles of interest are amplified by
Polymerase Chain Reaction (PCR). In practise the analyses of VNTR alleles at certain
loci can be used in e.g. forensic medicine, paternity testing, identification of unknown
persons and genealogy. In Fig. DNA / PCR.4, profiles are shown of paternal and maternal
DNA, (top of the figure) and the profiles of their four children (bottom of that figure) after
PCR amplification.
4
II
P4a-IV: Biochemie - DNA-profile analysis
POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is used to amplify a specific fragment of DNA from
a complex mixture of starting material, usually termed template DNA. This method does
require some knowledge of the DNA sequence information which flanks the fragment of
DNA to be amplified (target DNA). From this information two oligonucleotide primers are
chemically synthesized, each complementary to a flanking stretch at the 3’-end of the
target DNA, one oligonucleotide for each of the two DNA strands (Fig. DNA / PCR.5).
Fig. DNA / PCR.4. Representation of DNA profiles after PCR amplification. On top the gel pattern of
father and mother, at the bottom of this figure DNA profiles, from left to right of again father, four children
and mother, respectively.
This technique is analogous to the DNA replication process that takes place in cells, since
the outcome is the same, generation of new complementary DNA stretches based upon
existing ones. It is also a technique that has replaced, in many cases, traditional cloning
methods, since it fulfils the same function, the production of large amounts of DNA from
limited starting material. This is achieved, however, in a fraction of the time needed to
clone a DNA fragment.
Fig. DNA / PCR.5. Amplifying a specific DNA segment with a polymerase chain reaction. DNA
strands are separated by heating, then annealed to an excess of short synthetic primers (5’  3’) that flank
the region to be amplified. After polymerization, this process can be repeated many times.
P4a-IV: Biochemie - DNA-profile analysis
5
The requirements for PCR include: a DNA template for the polymerase to copy, short
(20 – 30 nt) DNA molecules to serve as starting points or primers for DNA synthesis, a
thermostable DNA polymerase to synthesize DNA, the four deoxyribonucleoside
triphosphates (dNTPs), and suitable reaction conditions to ensure good synthesis by the
DNA polymerase. PCR is performed using a thermostable DNA polymerase known as
Taq polymerase, which is isolated from Thermus aquaticus, a microorganism that inhabits
hot Springs in Yellowstone National Park. In the reactions you will set up today, the
template will be your own buccal cell DNA. The primers, the short DNA molecules that
function as starting sites for the DNA polymerase to begin synthesis, are specific for the
chromosomal region being amplified, D1S80. This is a locus on chromosome 1.
Stages in the PCR
A basic PCR cycle is composed of three reactions, all of which are performed at different
temperatures (Fig. DNA / PCR.6). In the first reaction (denaturation), the template DNA is
denatured to single strands at temperatures well above 90ºC for a short period of time. In
the second reaction (annealing), the temperature is lowered to about 65ºC to allow the
primers to anneal to the template DNA. And in the third reaction (extension) the
temperature is raised to the optimal temperature for the DNA polymerase to synthesize
DNA, i.e. 72ºC.
Fig. DNA / PCR.6. A simplified scheme of one PCR cycle that involves denaturation, annealing and
extension, ds: double stranded.
DNA synthesis proceeds from both of the primers until the new strands have been
extended along and beyond the target DNA they will contain a region near their 3’ ends
that is complementary to the other primer. Thus if another round of DNA synthesis is
allowed to take place, not only the original strands will be used as templates but also new
strands. Most interestingly, the products obtained from the new strands will have a precise
length, delimited exactly by the two regions complementary to the primers. As the system
is taken through successive cycles of denaturation, annealing and extension (Fig. DNA /
PCR.6.) all the new strands will act as templates and so there will be an exponential
increase in the amount of DNA produced. The net effect is to selectively amplify the target
DNA flanked by the primers.
6
III
P4a-IV: Biochemie - DNA-profile analysis
PCR AMPLIFICATION OF THE D1S80 LOCUS IN HUMANS
DNA Profile Analysis
3.1 Introduction
The D1S80 (Kasai et al. 1991) locus is a Variable Number of Tandem Repeat region
located on chromosome 1 in humans. At this locus, people have between 14 and 41 copies
of a 16 base pair sequence, this is the basic repeat unit. Twenty eight alleles are known to
occur in humans. These alleles consist of different numbers of repeats of the 16 base pair
sequence. Because over 80 % of the human population is heterozygous at this locus, that
means they have two different alleles, this locus is commonly used for identification
purposes.
3.2 Methods
Determining the number of tandem repeats requires three general steps:
1. Obtain a sample of human chromosomal DNA.
2. Make many copies of specific VNTR DNA from that sample.
3. Analyse the amplified VNTR DNA fragments.
The primers used in this experiment bracket the D1S80 locus and selectively amplify that
region on chromosome 1. Following PCR amplification, alleles will be separated according
to size using Agarose gel electrophoresis. After staining with ethidium bromide, one or two
bands will be visible in each lane, indicating whether an individual is homozygous or
heterozygous for the D1S80 locus. Different alleles appear as distinct bands, each
composed of several billion copies of the amplified allele. The position of a band in the gel
indicates the size (and the number of repeat units) of a D1S80 allele: smaller alleles move
a longer distance from their origin, while larger alleles move a shorter distance in the
agarose gel.
3.3 Procedure
3.3.1 Obtaining the DNA Sample
The following procedure is a modified version of that described by Walsh et al. (1991).
1. Vigorously scrape the inside of the cheek with a sterile toothpick. Break the
toothpick and place the end with the cells in a sterile 1.7 ml microcentrifuge tube.
2. Add 1 ml of sterile H2O, vortex the sample for 10 seconds, and spin in a microcentrifuge for 45 seconds at maximum speed.
3. Remove the piece of toothpick with sterile forceps. Rinse the tip of the toothpick in
the upper part of the solution and spin again. A ”clear” pellet of cells must be
visible. If not the scraping must be repeated.
4. Remove 970 µl H2O without disturbing the pellet. (With a 1 ml pipette, 800 µl and
with a 200 µl pipette, 170 µl of the supernatant is taken off, just to be sure not to
take away the cell pellet!)
5. Add 200 µl H2O and add a little amount of Chelex–100 to the sample with a sterile
yellow tip.
6. Vortex the sample for 10 seconds.
P4a-IV: Biochemie - DNA-profile analysis
7
7. Add 2 µl of Proteinase K solution to the sample and agitate.
8. Incubate at 56ºC for 5 minutes.
9. Vortex the sample for 10 seconds.
10. Incubate in a boiling water bath for 8 minutes.
11. Vortex the sample for 10 seconds.
12. Spin in a microcentrifuge at maximum speed for 3 minutes.
There should be two distinct layers visible in the reaction tube after completion of step 12.
The bottom layer contains the Chelex and cellular debris, while the top layer is an aqueous
solution containing the chromosomal DNA.
13. Transfer 150 µl of the supernatant to a fresh tube. Don’t transfer some of the
Chelex.
The sample is now ready for PCR amplification or may be stored temporarily at 4 ºC or
frozen at –20 ºC. To reuse these samples after storage, thaw if necessary and repeat steps
11 to 13.
3.3.2 PCR Amplification of the D1S80 VNTR Locus
1. For each PCR reaction the following components are added to a 200 µl
microcentrifuge tube (on ice).
o
25 µl PCR Mix (contains dNTP’s , Taq DNA polymerase, PCR buffer and
Mg2+ ions)
o
2 µl 20 µM Primer VNTR1 (sequence: 5' gaaactggcctccaaacactgcccgccg 3')
o
2 µl, 20 µM Primer VNTR 2 (sequence: 5' gtcttgttggagatgcacgtgccccttg 3')
o
20 µl DNA solution (obtained in 3.3.1)
2. Mix the solution and spin briefly.
3. The reaction tubes are placed into a thermal cycling machine programmed for 35
cycles with the following parameters.
First, heat the reaction mixture at 94ºC for 2 minutes, then 35 cycles:
o
o
o
Denaturation
Annealing
Extension
94 ºC for 30 secs
65 ºC for 30 secs
72 ºC for 1 minute
At the end, heat the reaction mixture for 2 minutes at 72 ºC.
4. Completed reactions may be stored at -20ºC indefinitely.
3.3.3 Preparation of an Agarose Gel
Agarose gel electrophoresis is a fast and convenient way to determine the quantity, quality
and length of DNA. By choosing the appropriate agarose concentration (0.3 – 2%),
molecules from 100 bp to 20 kb can be separated by length. Smaller molecules can be
fractionated on polyacrylamide gels, larger molecules (even complete chromosomes) by
field–inversion gel electrophoresis. For a complete description of these techniques, we
refer to the textbooks.
8
P4a-IV: Biochemie - DNA-profile analysis
Alternatively, separated DNA fragments can be transferred to nitrocellulose or nylon
membranes (Southern blotting) and detected by hybridization with a labelled probe; this
increases the sensitivity to the pg range, (pg = 10-12g).
Although originally an analytical technique, it is possible to isolate DNA fragments from
agarose gels, suitable for further manipulations.
Here, we will pour an agarose gel, which we will use to analyze the PCR fragments
prepared in the previous experiment (3.3.2).
NOTE:
We have chosen the TBE concentration as low as possible to lower the
conductivity and the heat dissipation, allowing the use of higher voltages.
1.
Prepare an agarose gel with a thickness of about 0.6 cm and a concentration of 2.0 %
(w/v) agarose in 0.5 x TBE buffer.
2.
Clean the gel trays, the barriers and the combs with distilled water and ethanol.
3.
Place the tray horizontally (Fig. DNA / PCR.7) and position a barrier at both ends of
the tray. The comb will be positioned when the agarose is in the tray (and while it is
still liquid).
Fig. DNA / PCR.7. Agarose gel electrophoresis tray and position of comb in agarose
4.
Solubilize agarose in buffer in a microwave oven. When completely homogeneous,
use about 1 ml of hot agarose solution to fix the barriers in their positions, and allow
the rest of the agarose to cool down to approx. 65°C. During cooling, the solution has
to be swirled to keep it homogeneous.
5.
Below 65°C, the ethidium bromide (10 mg/ml) can be added to the agarose solution
to a final concentration of 0.5 g/ml. Swirl to mix, but avoid air bubbles.
CAUTION:
6.
Ethidium bromide is a carcinogen. Handle carefully and wear gloves.
Pour the agarose/ethidium bromide solution into the gel tray and position a comb
vertically near one edge of the tray. Check for air bubbles trapped by the teeth of the
comb.
NOTE:
If the comb touches the tray, your sample will leak away.
7.
Let the gel harden until it becomes milky and opaque (approximately 30 min).
8.
Pour TBE (0.5 x) electrophoresis buffer on the gel and gently remove the barriers.
The gel should be totally submerged in buffer, but not covered by more than 0.5 cm.
This is often designated as a 'submarine' gel to distinguish it from other gel types.
9.
Remove the combs gently and check for air bubbles in the slots.
NOTE:
If you remove the comb too early, the slots will be filled with fluid agarose.
P4a-IV: Biochemie - DNA-profile analysis
9
3.3.4 Separation of DNA Fragments Using Agarose Gel Electrophoresis
Once the PCR reaction is completed, the DNA is ready for analysis using gel
electrophoresis.
1.
Add 5 µl of loading mix to each PCR sample, mix well and put the samples into
adjacent wells on the gel.
2.
A DNA ladder of 50 bp is used. Take 5 µl, add 15 µl TE buffer and 2 µl of load mix.
CAUTION:
3.
Don't contaminate loading mix with DNA!
Pipet 15 l of the samples carefully in the slots. Use adjacent slots, but don't use the
outer slots for the markers. Keep extreme end of tip steady at the rim of the slots and
empty the pipet slowly, avoiding air bubble formation. Be careful not to puncture the
bottom of the well, this will cause leakage of the sample. Note down which slot
contains which sample!
NOTE:
If you cannot easily see the slots, slip a dark sheet of paper or plastic under the
gel tray.
4.
Connect cables in the right way. (What is the charge of the DNA fragments?)
5.
Electrophoresis is done at 100 V constant till the bromophenol band is migrated out
of the gel. Note the change in current during electrophoresis. After the run, check the
pH at the anode and the cathode with a piece of pH-paper (pH 6 – pH 10, be careful,
buffer contains some Ethidium bromide!).
NOTE:
A low voltage gradient (1-2 V/cm) is recommended for the separation of large
fragments (> 10 kb); small fragments can run much faster without losing their
resolution.
6.
Put on gloves and allow buffer to drain away by tilting the tray, but keep a finger
against the gel to avoid slipping away. Dry with a tissue. (Be careful, gel and buffer
still do contain Ethidium bromide).
7.
Inspect the gel on an UV transilluminator at 312 nm, protecting your eyes with UVsafety goggles. Make a picture of the gel, using the GelDoc system.
NOTE:
8.
Do not use UV light of 260 nm, as it will damage the DNA. UV light of 366 nm
gives a higher background by exciting ethidium molecules in the gel that are
not bound to DNA.
Clean the GelDoc system.
10
IV
P4a-IV: Biochemie - DNA-profile analysis
RESULTS AND DISCUSSION
1. Examine the photograph of the stained gel containing your sample and those from other
individuals. Orient the photograph with the sample wells at the top. First, ascertain whether
or not you can see a diffuse (fuzzy) band of "primer dimer", that appears at the same
position in each lane toward the bottom of the gel. Primer dimer is not amplified human
DNA, but is an artefact of the PCR reaction that results from primers amplifying
themselves. Excluding primer dimer, interpret the allele bands in each lane of the gel.
2. Population studies have identified about 28 different alleles at the D1S80 allele, and
estimate that 90% of the individuals are heterozygous at this locus. Determine the number
of different alleles represented among your classmates and the percent of heterozygous
individuals. How do the group data compare with that of general population? What reasons
can you give for the observed differences?
3. Based on your results, do you think this protocol could be used to link a suspect with a
crime or establish a paternity relationship? Why do you think so? How could you modify
the experiment to improve its ability to positively identify individuals?
4. Allele sizes can be estimated in agarose gels simply by comparing their positions to the
ladder of size markers included in one lane of the gel. However, a closer determination of
allele sizes can be obtained by graphing the function that determines the migration of
linear DNA fragments through an agarose gel:
D = 1 / log10 M
where D equals distance migrated and M equals the molecular mass of the fragment. For
simplicity's sake, biologists substitute base pair length for molecular weight in this
calculation.
Compare the largest and smallest allele observed in your group with the known range of
most D1S80 alleles, 369–801 base pairs.
5. Explain the results of your experiment when amplifying the VNTR using your DNA as a
template. What does this tell you about your genetic make-up concerning the VNTR? Are
you homozygous or heterozygous? Calculate the number of repeats in your alleles.
6. Assume that you have two copies of the VNTR in your genome sample. Hypothetically,
how many copies of the amplification product were made after 35 cycles of PCR?
7. What are the components of the PCR reaction mixture, and what are their roles during
the reaction?
8. Which process is occurring at each of the temperature during a PCR cycle?
9. What is Chelex–100 and what is its function in 3.3.1 point 5?
10. What is Proteinase K and what is its function in 3.3.1 point 7?
11. Why are the samples boiled for 8 minutes in 3.3.1 step 10?
12. Explain the change in pH during electrophoresis and which reactions take place at the
cathode (–) and at the anode (+)?
13. Where can you find the VNTR1 and VNTR2 primers in the DNA sequence of an allele
of human D1S80 given on page 12?
P4a-IV: Biochemie - DNA-profile analysis
11
Reagents and Equipment
–

Micropipettes of 20, 100, 200 and 1000 µl capacities.

Micropipette tips

Microcentrifuge tubes of 200 µl, 0.65 ml and 1.7 ml capacities.

Microcentrifuge

Sterile toothpicks

Proteinase K solution

Chelex – 100 resin, biotechnology grade (Sigma)

Vortex test tube mixer

PCR Master mix. This solution contains Taq polymerase, MgCl2 and
all the four dNTP (Promega).

PCR primers VNTR1 and VNTR2 for D1S80 locus, 20 µM each in
TE (see text for primer sequence).

Electrophoresis power supply

Ultraviolet illuminator

Agarose

0.5x TBE: (90 mM Tris, 90 mM H3BO3, 0.25 mM EDTA, pH 8.3).

TE buffer, 10 mM Tris/HCl, 1 mM EDTA pH 8.0

Ethidium bromide (10 mg/ml)

Gel loading buffer:
Loading mix, 11 x concentrated (-20°C):
5%ficoll
to avoid mixing with the TBE while pipetting the sample in the slot.
50%glycerol
0.25% Bromophenol blue (migrates as DNA of about 200 bp)
1.0% SDS (inactivates enzymes and stimulates their migration)
10 mM EDTA
50 mM Tris/HCl (pH 8.0).

DNA size marker: 50 bp marker.

Thermal cycler
12
P4a-IV: Biochemie - DNA-profile analysis
References
Kasai, K., Nakamura, Y. & White, R. (1990) Amplification of a Variable
Number of Tandem Repeats (VNTR) Locus (pMCT118) by Polymerase
Chain Reaction (PCR) and Its Application to Forensic Science. Journal of
Forensic Sciences, 35(5) 1196-1200.
Walsh, S., Metzger, D. & Higuchi, R. (1991) Chelex¬ 100 as a Medium for
Simple Extraction of DNA for PCR-Based Typing from Forensic Material.
BioTechniques, 10(4), 506-513.
DNA Sequence of an Allele of Human D1S80
DNA Flanking Sequence at 5’ end:
GGATTACCAT
CACTGCCCGC
TGACTCAGGA
AGATAAGCGC
GATGCCATTC GTTGGAAACT GGCCTCCAAA
CGTCCACGGC CGGCCGGTCC TGCGTGTGAA
GCGTATTCCC CACGCGCCAG CACTGCATTC
TGGCTCAGTG
followed by:
Tandem Repeats of 16 base sequence
TCAG –CCCAAGG -AAG
ACAGACCACAGGCAAG
GAGGACCACCGGAAAG
GAAGACCACCGGAAAG
GAAGACCACCGGAAAG
GAAGACCACAGGCAAG
GAGGACCACCGGAAAG
GAAGACCACCGGCAAG
GAGGACCACCGGCAAG
GAGGACCACCAGGAAG
GAGGACCACCAGCAAG
GAGGACCACCAGCAAG
GAGGACCACCAGGAAG
GAGGACCACCAGGAAG
GAGGACCACCGGCAAG
GAGGACCACCAGGAAG
GAGGACCACCAGGAAG
GAGGACCACCGGCAAG
GAGGACCACCAGGAAG
GAGAACCACCAGGAAG
GAGGACCACCAGGAAG
GAGGACCACCAGGAAG
GAGGACCACTGGCAAG
GAAGACCACCGGCAAG
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
followed by
DNA Flanking Sequence at 3’ end:
CCTGCAAGGG GCACGTGCAT CTCCAACAAG ACGGCTTCGC
GGTCCATTTT
P4a-IV: Biochemie - DNA-profile analysis
13
D1S80 Allele Frequency (Literature)
no. of
Observed
repeats Observed Frequency Homozygotes
14
1
0.004
0
15
0
0.000
0
16
3
0.012
0
17
2
0.008
0
18
51
0.206
2
19
3
0.012
0
20
7
0.028
0
21
9
0.036
1
22
12
0.048
0
23
3
0.012
1
24
67
0.270
11
25
9
0.036
1
26
7
0.028
0
27
7
0.028
0
28
10
0.040
1
29
13
0.052
0
30
9
0.036
0
31
15
0.060
0
32
8
0.032
0
33
3
0.012
0
34
4
0.016
0
35
1
0.004
0
36
1
0.004
0
37
0
0.000
0
38
0
0.000
0
39
0
0.000
0
40
0
0.000
0
41
0
0.000
0
>41
3
0.012
0
TOTAL
248
17