DNA Typing
Introduction &Procedures
1. Identifying the specific individual who is the source of a sample of DNA
OR
2. Determining specific ‘characteristics’ of a sample of DNA
10/07/13
Various names: DNA fingerprinting, DNA profiling, DNA (gene) testing
I. History of DNA Typing
II. Testing for identity vs. Testing to determine specific alleles or characteristics
III. Many decisions must be made when testing DNA
IV. Procedure I: RFLP analysis
POST-TEST 1
V. Procedure II: PCR
VI. PCR combination STR Test
POST-TEST 2
Start Recorded Lecture Part 1 (~ 15 min.)
I. History
A. First cases utilizing DNA fingerprinting
1. Immigration -1985
2. Rape and murder
B. Immediate media blitz favoring DNA technology.
-Defense attorneys were caught off-guard.
-Juries, suspects, attorneys, and judges rushed to accept the results of DNA
testing.
-Over 100 cases occurred before...
1
C. First successful challenge to exclude the DNA evidence: murder
(Meaning there was DNA evidence, but the judge did not allow the jury to see it)
-The crime: police found a pregnant woman and her two-year old daughter
stabbed to death.
-Blood was found on the custodian’s watch.
-Testimony by a private DNA testing company, Lifecodes Corporation stated that
the blood on the watch matched the murdered woman’s.
-For the first time, the defense undertook a thorough examination of the genetic
analyses and mounted the first extended effort to have DNA evidence excluded.
-They successfully argued that…
1) Lifecodes did not use accepted scientific techniques.
2) Lifecodes did not follow their own procedures for interpreting their data.
-The DNA evidence was found inadmissible.
-Later that year, the custodian confessed and was found guilty.
D. Evaluation of DNA typing procedures.
1. 1989: National Association of Criminal Defense Lawyers set up a DNA Task
Force; headed by Barry Scheck and Peter Neufeld.
-Critical assessment of DNA typing procedures and analysis.
2. At the same time, the National Academy of Sciences also began a more
detailed look at the procedures. They issued a report that basically said...
a. The technology was accepted theoretically and for practical
applications.
b. But, standards for forensic use should be determined.
3. After over 400 technical papers, over 150 court cases, 100 scientific
conferences, three sets of DNA analysis guidelines, and a three year study by the
National Research Council, most attorneys recommended the continuation of
DNA typing.
Complete the Part 1 Recorded Lecture Self-Evaluation Questions.
2
Start Recorded Lecture Part 2 (~ 41 min.)
II. There is a variety of DNA typing procedures. Different kinds of procedures provide
different types of information. Labs can test for identity or test to only determine specific
alleles or characteristics of certain area of the DNA
Note:
Most DNA is the same from person to person.
Only about 0.1- 0.5% varies between individuals.
Some DNA typing procedures DO identify
Some DNA typing procedures DO NOT
the individual person who is the source of a identify the individual person who is the
sample of biological material.
source of a sample of biological material,
but can provide some information about
that person’s DNA.
No two individuals have the exact same
sequence of nitrogen bases on every
chromosome.
Information about an individual gene (the
exact alleles – the genotype- the person
inherited for one gene) will not identify an
individual.
Each person’s DNA is the same in every
cell.
However, these tests may provide useful
information, such as if a person has an
allele for genetic disorder.
An individual’s profile remains the same
throughout life.
Questions: What are somatic
cells? What is a ‘genome’?
3
III. Many decisions must be made when testing DNA.
A. In general, what area(s) of the DNA are tested?
When testing for identity, areas of the DNA are used that vary among individuals.
POLYMORPHISM:

DNA polymorphism:
o
DNA SEQUENCE POLYMORPHISM:
o
DNA LENGTH POLYMORPHISM:
4
B. Specifically, what ‘Genetic Markers’ will be used?
Genetic Marker: Any segment of DNA that is known, can be identified, and tested.
1. Genetic markers that are Length Polymorphisms: Variable Number Tandem
Repeats (VNTRs).
a. VNTRs: Sequences of DNA, 2 to ~80 (or more) bp, that are repeated (a
few times to thousands of times).
Most VNTRs are non-coding (don’t code for proteins).
Most is junk DNA.
There are many different VNTRs with different sequences
repeated.
VNTRs are scattered throughout the genome on all nuclear
chromosomes.
The number of repeats you have of a certain VNTR was inherited
from your parents.
This is the basis for RFLP DNA testing.
b. Two categories:
1) STRs: These are short sequences of nucleotides (example:
ATCG) that are repeated over and over again a number of times in
tandem.
2) LTRs: These are longer sequences of nucleotides that are
repeated over and over again a number of times in tandem.
5
2. Examples of Genetic markers that are Sequence Polymorphisms:
a. SNPs (Snips): These are single nucleotide polymorphisms in which one
nucleotide nitrogen base at a specific locus varies among individuals.
b. Sequencing larger sections of the DNA: This is the basis for
Mitochondrial DNA (mtDNA) testing.
“Mitochondrial DNA (mtDNA) typing is increasingly used in human identity testing
when biological evidence may be degraded or when quantities of the samples in question
are limited. In humans, mtDNA is inherited strictly from the mother. Consequently,
mtDNA analysis cannot discriminate between maternally related individuals (e.g., mother
and daughter, brother and sister). However, this unique characteristic of mtDNA is
beneficial for missing person cases when mtDNA samples can be compared to samples
provided by the maternal relative of the missing person.
For humans, the mtDNA genome is approximately 16,000 bases (A, T, G, and C) in
length containing a "control region" with two highly polymorphic regions. These two
regions, termed Hypervariable Region I (HV1) and Hypervariable Region II (HV2), are
342 and 268 base pairs (bp) in length, respectively, and are highly variable within the
human population. This sequence (the specific order of bases along a DNA strand)
variability in either region provides an attractive target for forensic identification studies.
Therefore, sequencing of these areas is usually performed.”
Complete the Part 2 Recorded Lecture Self-Evaluation Questions.
6
Start Recorded Lecture Part 3 (~ 20 min.)
IV. Procedure I: RFLP analysis
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
(Discovered by Alec Jeffreys.)
This procedure may determine identity of cells, usually with an
individual test.
All 46 nuclear chromosomes are typically utilized in this procedure.
Restriction
Fragment
Length Polymorphism
This refers to the use of
restriction enzymes to cut
the human DNA at
restriction sites creating
restriction fragments.
Each person contains a
specific recognition sequence
in many places in the DNA.
Polymorphism= any
variation between individuals.
Restriction sites are specific
sequences, usually around 6
nucleotides long.
A.
If all 46 chromosomes are
exposed to a restriction
enzyme, the DNA will be cut
into a number of differently
sized restriction fragments.
When cut with the same
restriction enzyme(s),
different people will produce
differently sized fragments
(the number of nucleotides in
each fragment will be
different).
An example of a restriction site is the one recognized by the restriction
enzyme EcoRI.
This enzyme cuts the following sequence:
GAATTC
CTTAAG
7
Each person's DNA is unique. Although everyone has the same restriction sites
(sequences of nitrogen bases) in many areas of their DNA, they may be in
different places than in other people's DNA.
Therefore, two different people, whose DNA is cut with the same restriction enzyme, will
end up with restriction fragments (stretches of DNA produced by restriction enzyme
cutting) of different lengths (RFLPs).
Length polymorphism:
A. VNTRs are used in the RFLP procedure
VARIABLE NUMBER TANDEM REPEATS
Complete the Part 3 Recorded Lecture Self-Evaluation Questions.
Start Recorded Lecture Part 4 (~ 17 min.)
C. Jeffries combined two discoveries: 1) Restriction enzymes that will cut DNA at
specific restriction sites in the DNA and 2) VNTRs in human DNA.
8
2. A restriction enzyme that cuts DNA not within the VNTR but
on either side of the VNTR is selected.
Because different people have VNTRs with different numbers of repeats,
fragments of different lengths will be produced.
9
3. When all 46 chromosomes of an individual are exposed to a restriction
enzyme, the enzyme will cut the DNA anywhere the restriction site is located.
The result may be tens of thousands of restriction fragments of many different
sizes, from very large to very small (and many in-between). Each person will
have their own unique set of restriction fragments, from the longest to the
shortest.
4. Restriction sites are found in many places, not only near VNTRs. The
RFLP procedure eventually will be able to ignore all other fragments
and detect only the fragments containing VNTRS.
Complete the Part 4 Self-Evaluation Questions.
Start the Part 5 Recorded Lecture (~20 min.)
So the process of RFLP analysis first requires that you have a sample of DNA
that is then “cut up” (digested) in the lab using one or more restriction
enzymes.
The DNA is cut into fragments, now what? It’s still in a test tube and you
can’t tell one fragment from the next.
A way of separating the different sized fragments is needed.
D. RFLPs are then separated using GEL SLABS.
Gels have microscopic pores through which the restriction fragments move when
an electric current is applied. DNA has a negative charge, so the fragments travel
toward the positive pole.
Smaller fragments can more easily move through the pores, so they travel from
one end of the gel to the other faster than larger fragments.
Therefore, if all of the DNA fragments are placed at one end of the gel (the
origin) and the electric current is applied for a certain period of time, the smaller
fragments will have moved farther down the gel while the larger fragments are
closer to the origin.
10
The purpose of gel electrophoresis is to separate the different sized fragments.
At this point the VNTR fragments cannot be distinguished from other fragments.
Sample Questions:
Does DNA have a negative or positive charge?
During electrophoresis, will DNA move toward the positive or negative pole?
Which moves faster through the gel, larger or smaller restriction fragments?
One restriction fragment (restriction fragment #1) has 2070 nucleotides. Restriction
fragment #2 has 682 nucleotides. After 4 hours of electrophoresis, which restriction
fragment will be closer to the positive pole? Why?
Complete the Part 5 Recorded Lecture Self-Evaluation Questions.
11
Start Recorded Lecture Part 6 (~21 min.)
So now there are restriction fragments from the top of each gel lane to the bottom.
E.
After gel electrophoresis, the DNA is unzipped.
F.
Southern blotting: A sheet of nylon (the membrane) is placed over the
gel. The single-stranded DNA is transferred to the nylon membrane. This
is called Southern blotting. Southern blotting immobilizes and stabilizes
the DNA.
G.
How are the VNTR fragments differentiated from the other fragments?
In a different part of the lab, other single-stranded pieces of DNA
synthesized or obtained in the lab are used next. These small sections of
DNA are called PROBES.
-Probes have a specific sequence of nucleotides. A probe can have almost
any nucleotide sequence a researcher desires.
1. A single-stranded probe can bond to complementary sequences in
another sample of single-stranded DNA.
2. For example, a probe might have the sequence GTTTGA. If the probe
is combined with a person's single-stranded DNA, it will bind to all areas
of the DNA with the sequence CAAACT.
3.
Uses of probes- Probes bond to specific DNA sequences.
a. Some probes may have the sequence complementary to a
specific allele of a gene. For example, if you want to test for
cystic fibrosis, an allele complementary to it can be
synthesized.
b. In the RFLP procedure, probes are designed with a
complementary sequence to a tandem repeat and will,
therefore, bond to the fragments with the repeats.
4.
Probes can be radioactively labeled.
This means that a radioisotope (radioactive atom or molecule,
usually 32P) is attached to the probe.
12
H.
Autoradiography: If a probe has hybridized (bonded) with a
complementary sequence in the DNA being examined, it can be viewed
after it has been exposed to x-ray film.
Larger fragments
Dark bands indicate
fragments where
the probe has bound.
Smaller fragments
Autoradiograph (autorad). The dark bands indicate
areas where the radioactive probes have bound.
Different individuals have the complementary probe
sequence on different sized fragments.
Complete the Part 6 Recorded Lecture Self-Evaluation Questions.
13
Start the Part 7 Recorded Lecture (~10 min.)
I.
Example: Paternity testing
Each individual receives one set of chromosomes from their mother and
another, homologous set, from their father. Therefore, each parent
contributes roughly half of an individual's restriction fragments. The
mother's, baby's, and potential father(s)’s banding patterns are determined
and compared.
Consider the examples on the sample autorad.
Question:
How much of their DNA do parents give to their offspring?
Restriction Digestion and Electrophoresis Animations: Go to the site below and click on
play. http://www.dnalc.org/ddnalc/resources/animations.html
14
Overview: Steps of RFLP
Complete the Part 7 Recorded Lecture Self-Evaluation Questions.
15
16
DNA Typing Introduction and Procedures
POST-TEST 1
1.
Some definitions are given below. Write the appropriate word for each of the
definitions below.
a. Process of transferring DNA from the gel (after electrophoresis) to a nylon membrane;
this immobilizes the DNA in the positions they are in at the end of electrophoresis.
b. Process of moving molecules when an electric current is applied; the DNA (with a
negative charge) moves toward the positive pole; during RFLP analysis of DNA, this
separates the different sized fragments because smaller fragments move faster through
the gel.
c. All cells that make up the body except for sex cells; these are “body” cells.
d. A VNTR consisting of repeated segments with shorter repeated segments.
e. Areas in the DNA consisting of repeated sequences, these repeats are located adjacent
to one another; usually consist of non-coding DNA; often used in DNA testing.
f. A DNA marker consisting of single nucleotide differences between individuals.
g. All of the DNA in the cell, organism, species, or population.
h. Type of DNA testing procedure relying on length polymorphisms resulting from the
digestion of the DNA with restriction enzymes into different sized restriction fragments;
the fragments are separated during electrophoresis and transferred to a nylon membrane
during Southern blotting; a radioactive probe is applied to the DNA and autoradiography
shows the positions of the fragments where probe has bound.
17
i. The process of breaking the bonds between the nitrogen bases in the DNA double helix.
This results in unbound nucleotides that can bond to other unbound nitrogen bases.
j. Exposing a sample of DNA that has been bound to a radioactive probe to an x-ray film.
Portions of the DNA that are complementary to the probe will be viewed on the film as
"bands".
k. Any variation between individuals.
l. A radioactive atom or molecule; often 32P, used to "label" molecules called probes.
m. A restriction enzyme with the restriction site GAATTC.
n. Variation between individuals in the number of nucleotides in DNA fragments
produced after digestion with restriction enzymes.
o. A solidified substance composed of agar with pores throughout; used during
electrophoretic procedures.
p. A single-stranded sequence of nucleotides used to target and identify specific areas of
the DNA during DNA typing. They bond to specific single-stranded areas in the sample
DNA being tested. In the RFLP procedure, they are radioactively labeled and may be
complementary to VNTR sequences.
q. Any of several procedures used in forensics to either determine identity of cells or
specific characteristics of the sample DNA.
18
r. An enzyme which catalyzes chemical reactions which "cut" DNA at specific locations,
producing DNA fragments of varying sizes.
s. Any area of the DNA with variations in the nitrogen base sequence.
t. The specific sequence of nitrogen bases that a specific restriction enzyme recognizes
and "cuts".
u. Areas of the DNA that are known, and can be identified and tested.
2. What people have the same exact sequence of DNA nucleotides in their DNA?
3. All cells of the body have the same DNA complement (number of chromosomes)
EXCEPT…
4. Explain the reasons for your answer to #3.
5. Most DNA is different from person to person.
a. True
b. False
6. Approximately what percentage of the DNA is different from person to person?
7. How much of the DNA in ONE cell is required for RFLP analysis?
19
8. Explain your answer to #7. Why do you need this amount of the DNA?
9. What is meant by "identity" of cells?
10. Describe the different types of VNTRs.
11. List 3 names for DNA typing.
12. DNA typing theory and techniques are not currently accepted by defense attorneys.
a. True
b. False
13. RFLP: What is each letter an abbreviation for?
a. R:
b. F:
c. L:
d. P:
14. Can RFLP prove identity of cells? Explain your answer.
20
15. Who discovered the RFLP procedure?
16. What is the restriction site of EcoRI and where does this restriction enzyme cut the
site? Show the ends of the resulting fragments.
17. How many fragments will be produced if there are 5 restriction sites on a particular
chromosome that is digested with a certain restriction enzyme?
18. Which of the following are true concerning VNTRs?
a. The number of repeats in a person's VNTRs is inherited from the parents.
b. Most VNTRs are junk DNA.
c. Humans have only one VNTR sequence.
d. VNTRs are only found on chromosomes 5 and 15.
e. Each VNTR is repeated millions of times.
19. Read the following information.
Mother:
-Maternal chromosome 3 = VNTR repeated 5X
-Paternal chromosome 3 = VNTR repeated 8X
Father:
-Maternal chromosome 3 = VNTR repeated 16X
-Paternal chromosome 3 = VNTR repeated 12X
What are the possible VNTR patterns that a child of theirs might contain?
Maternal:
Paternal:
Maternal:
Paternal:
Maternal:
Paternal:
Maternal:
Paternal:
21
20. Where are the restriction sites that are used to create length polymorphisms in the
RFLP procedure?
21. Can a VNTR with restriction sites between repeats be used for RFLP analysis? Why
or why not?
22. During electrophoresis… Indicate all that apply.
a. gels are used with microscopic pores (tunnels) through which the DNA moves.
b. DNA is positively charged, so it will move toward the negative pole.
c. Larger fragments are bigger so they move faster than smaller fragments.
d. After 4 hours, a fragment with 10 repeats will be closer to the positive pole than
a fragment with 14 repeats.
23. The end of the gel where the DNA is loaded is called the…
24. What is meant by the size of a DNA fragment?
25. What is the purpose of electrophoresis?
26. A way of transferring the DNA to a more stable substance that would keep the
fragments in their relative positions is required. What is this procedure called?
27. After electrophoresis, the DNA fragments are found in all sizes, from the smallest to
the largest (the restriction sites are found in many places other than on either side of the
VNTRs). How can the technician find the VNTRs among all of the fragments and
pinpoint its position in the gel (which will tell the technician the size of the fragment)?
22
28. The radioactive areas are "seen" by placing the membrane against an x-ray film. This
procedure is called…
29. After the above procedure, the film is called an…
30. The dark areas on the film where the fragments with VNTRs are located are called…
33. Why don't scientists prefer the term DNA "fingerprinting"?
34. Describe how testing for identity is different from testing for specific characteristics
or alleles of the DNA.
35. Why, theoretically, is it possible for some DNA typing procedures to definitely prove
identity of cells?
36. When testing for identity, what qualities should the DNA possess?
37. Describe the immigration situation and the rape/murders that were the first cases
utilizing DNA typing.
23
38. Describe the first successful case where DNA typing was excluded from the court
proceedings.
39. Describe major types of markers used for identity testing.
40. Describe how VNTRs are used to create length polymorphisms, in detail.
41. Describe mtDNA and the areas of the mtDNA that is tested.
42. Know the steps of RFLP in order, their purpose, and how they are performed.
24
Start the Part 8 Recorded Lecture (~27 min.)
V. Procedure: PCR
A. The polymerase chain reaction (PCR) mimics DNA replication in a cell.
B. The process of PCR will make copies of a specific sequence of DNA in a test
tube. PCR also is called DNA amplification.
C. Kary Mullis invented the procedure in 1984 and was awarded the Nobel Prize
in 1994. This is extremely unusual. Generally the Nobel Prize is not given until
after a lifetime of research, rarely for the invention of one technique. The
procedure is so useful that it has been referenced in over tens of thousands of
scientific publications.
D. Why is it so useful?
1.
In order to detect and test, a certain number of copies of the DNA
must be present. At 100% efficiency, PCR can produce over one
billion copies of the DNA in a few hours. The copies can then be
used for DNA typing, sequencing, etc. PCR is used to make
enough DNA to detect and test. It also may even be incorporated
as part of the test itself.
2.
In theory, PCR is so sensitive that only one copy of the DNA is
required. RFLP, on the other hand requires a much greater amount
of DNA, roughly that found in a dime/quarter-sized bloodstain.
3.
In addition, PCR targets a specific sequence, not many different
areas of the DNA. This simplifies the study of individual genes or
loci without having to wade through all three billion+ base pairs of
human DNA.
4.
It also makes it possible to test highly degraded DNA since only
relatively small segments are needed. RFLP analysis requires
fairly intact DNA.
E. Disadvantages: Is PCR too good? Because PCR is so sensitive, contamination
is an ever-present concern.
25
F. The specific sequences of DNA on both sides of the target sequence (the DNA
being amplified) are required before PCR can be performed.
The sequences adjacent to both ends of the target DNA are called the
flanking sequences.
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
If the flanking sequences are known, researchers can synthesize singlestranded DNA complementary to the flanking sequences for use as
Individual
nucleotides
26
G. Basic materials and equipment:
1. Target DNA and flanking sequences (at least one copy)
2. DNA polymerase: enzyme utilized by and isolated from living cells for
DNA replication
3. An ample supply of all four DNA nucleotides (A, T, C, G)
4. Large supply of two different single-stranded primers: sequences of
DNA, ~20-30 nucleotides
-One primer is complementary to the flanking sequence on one
side of the target DNA
-The other primer is complementary to the flanking sequence on
the other side of the target DNA
-The primers are complementary to opposite strands of the target
DNA
5. A machine called a thermocycler, which holds the tubes
containing the above materials, is necessary to cycle the temperature at
various points during the procedure.
PCR STEPS
27
Cycle One
a. Heat mixture in tube to 90-95°C (194-203F) to unzip the DNA (~30 seconds to
several minutes).
b. Cool mixture to 50-65°C (122149F) to allow the single-stranded
primers to bond to the unzipped
flanking DNA (20 seconds to one
minute).
c. Heat mixture to 72-75°C (161.6-167F) allowing the
DNA polymerase to synthesize new DNA
complementary to the target DNA (1-several
minutes).
After this first cycle, there are now two copies.
This same cycle is repeated with the two copies.
Each cycle doubles the number of copies of the target sequence.
Polymerase Chain Reaction Animation: Go to the link below and click on play.
http://www.dnalc.org/ddnalc/resources/animations.html
Depending on the sequence(s) amplified, the DNA can be tested for variations
between individuals using a variety of DNA typing techniques.
Complete the Part 8 Recorded Lecture Self-Evaluation Questions.
28
Start the Part 9 Recorded Lecture (~42 min.)
VI. PCR Combination Test: STR Testing (Short Tandem Repeats)
A. STRs are short sequences of DNA that are repeated a variable number of
times.
For example, the entire STR 16 bp sequence of "gatagatagatagata"
represents 4 copies of the repeat "gata". Tetranucleotides (repeats
consisting of 4 nucleotides) are commonly amplified for use in forensics.
LTR/RFLP test.
Number of base ~30 to ~80.
pairs per repeat
PCR/STR test.
2-5.
Size of each
individual
fragment (all
repeats) in bp
Can be thousands….
~100-350 bp.
Amplifiable?
No.
Yes.
Size of sample
required
Restriction
digestion
Large, cannot be highly degraded.
Small, can be highly degraded.
Yes.
Electrophoresis
Yes.
No
(PCR primers determine the
fragment).
Yes.
Variability
Highly polymorphic. Only one
individual test usually required.
29
Least amount of variability.
Several STRs usually tested at
one time for identification
purposes.
C. Using STRs in DNA identification
1. The concepts are basically the same as in RFLP, but the fragments are of
shorter length – smaller number of bp in each repeat and a smaller range in repeat
number.
2. DNA identification generally involves simultaneous co-amplification of several
different STR sequences. These are called multiplex systems. All of the required
primers, for each STR, are included in one tube for PCR.
3. After amplification, electrophoresis is performed.
-All of the amplified STRs are run together in one lane.
-Electrophoresis can be accomplished in two ways:
a. Using gel slabs (See next page.)
b. Using capillaries (p.34)
30
a. Using gel slabs: The sample may then be electrophoresed on a gel, thereby separating
the different sized fragments (based on number of repeats).
1) The table below lists the STRs in three different systems.
System 1: CTTv
CSF1PO
Names of TPOX
different
THO1
STRs
vWA
System 2: FFFL
F13A01
FESFPS
F13B
LPL
System 3:
GammaSTR
D16
D7
D13
D5
2) Below is a picture of actual gels with samples taken from six different people.
The
numbers on
the right
represent
the number
of copies of
the repeat.
The alleles
of a
particular
STR are
symbolized
by the
number
repeats.
Lanes 1-6 are the six different people.
L is a “Ladder” of DNA containing fragments with known repeat numbers (different alleles) for each of the
STRs.
The STR sequences chosen here have a limited number of known repeats that do not overlap.
Therefore, all bands may be seen, and the STR for each band can be determined.
31
3) The link below is a database containing detailed information on each STR
along with important information concerning STRs and their use.
Example: System 1: CSF1PO STR
Click here: STR Internet Database
From the above site:
Click on “STR Fact Sheets”.
Click on “CSF1PO”
Determine the chromosome where this STR is found:
Determine the range in the number of possible repeats:
Repeat sequence:
4) How are the bands seen?
Each amplified copy of the target DNA (an STR) remains attached to the primers.
a) The primers used during the PCR process can be labeled (or ‘tagged’) with
radioactivity (a radioisotope was attached to each primer as it was made). Since each
PCR product has the primers attached, each fragment will be radioactive and can be
detected after Southern blotting and autoradiography. The primers are functioning as a
probe, also.
b) Primers used during PCR to amplify the STRs can be ‘tagged’ with a fluorescent dye
(Click here: What is Fluorescence) instead of radioactivity. Since primers are attached to
each amplified copy of the target DNA, each STR (DNA fragment) will have a
fluorescent tag. When the gel is run, the colors are detected with a fluorescent scanner
(lasers in the scanners activate the dyes). Data collection occurs in "real time" as
fluorescently labeled fragments migrate past the detector at a fixed point and viewed as
they are collected. If different colored dyes are used, even overlapping STRs can be
tested together.
32
5) Electropherogram: A graphical representation illustrating discrete DNA
fragments.
Electropherograms are produced by a scanner moving across the electrophoresed sample
(or, likewise, the sample moving by the scanner). The scanner picks up the dyed DNA,
and the data is plotted on a computer. The peaks are roughly analogous to bands on a gel.
Example of STR electropherogram.
33
b. Using Capillary Electrophoresis instead of gel slabs
1) As the name implies, capillary electrophoresis (CE) is carried out in a
microcapillary tube rather than in a gel slab.
2) A CE system provides automated gel assembly, sample loading,
electrophoresis, detection, and analysis. Once samples, gel and buffer are loaded
onto the instrument, the capillary is filled with gel, and the sample is loaded
automatically. Up to ninety-six samples are automatically loaded and analyzed
unattended.
3) Since the CE is fully automated, the chance of human error in the postamplification handling and analysis of samples can be reduced, a significant
benefit when large numbers of samples are processed.
4) CE is carried out at a much higher voltage (It’s ok for the gel to melt!).
Therefore, separation of fragments from a single sample is completed in twenty
minutes or less.
Click here for Capillary Electrophoresis Animation (Link is often down)
34
C. Comparing STR/Gel Slabs and STR/Capillary Electrophoreis
STR/Gel Slabs
STR/Capillary
Electrophoreis
1. Extract the DNA from the cells.
2. PCR:
Amplify the STR sites
(Co-amplify all sites in
multiplex systems).
3. Electrophoresis
separating the different
sized STR fragments.
The STRs are
electrophoresed with
“ladders” of known alleles
with specific repeat
numbers.
4. View the DNA Profile
5. Analysis
6. Automation
Use primers “tagged” with
Use primers “tagged” a dye
radioactivity or a dye
(fluorescent is common).
(fluorescent is common).
This labels the sequences
This labels the sequences
for viewing. A different set
for viewing. A different set of ‘probes’ is not required.
of ‘probes’ is not required.
The primers act as probes.
If multiplex systems are used and the primers are
fluorescently tagged, use different color fluorescence for
each primer set.
Electrophorese the samples Electrophorese the samples
in a gel separating the
in a microcapillary tube
different sized fragments.
If primers were radioactive:
fragments must be nonoverlapping between STR
systems. If fluorescently
tagged
If primers were radioactive:
Southern blotting &
Autoradiography. If
fluorescently tagged
If using radioactivity, an
autorad: Fragment position
is determined and compared
to the ladder. If
fluorescently tagged
Can only be partially
automated.
35
Fragments can be
overlapping since different
colors are used for each
STR.
Fluorescent primers: The
samples are scanned as the
fragments are
electrophoresed.
Electropherogram: Peak
color and position are
compared to a known
ladder.
Can be almost completely
automated.
Overview: Steps PCR/STR
Complete the Part 9 Recorded Lecture Self-Evaluation Questions.
36
DNA Typing Introduction and Procedures
POST-TEST 2
1.
Some definitions are given below. Write the appropriate word for each of the
definitions below.
a. Graphical representation illustrating DNA fragments, sequences or nucleotide bases as
peaks. Each peak is roughly analogous to a band on a gel.
b. Procedure that produces copies of a sequence of DNA. The number of copies doubles
with each cycle.
c. Areas of the DNA that are copied during PCR.
d. Machine that cycles the temperature during PCR.
e. Short sequences of DNA complementary to opposite strands flanking the target DNA
to be amplified during PCR. They are synthesized in the lab.
f. Enzyme used by cells for DNA replication and by labs during the PCR process to
synthesize new copies of the target sequence.
g. A condition of having two of the same alleles for a particular gene.
h. A condition of having two different alleles for a particular gene.
37
i. What specifically is found in each of the copies produced by PCR?
j. Repeated sequences with 2-5 base pairs per repeat. Each fragment consists of ~100350 base pairs.
k. Many STRs co-amplified and tested simultaneously are called
systems.
l. What is the order of the nitrogen bases in the STR CSF1PO?
m. Electrophoretic procedure carried out in a microcapillary tube.
n. Sequences of DNA on either side of the target DNA. During PCR, synthesized DNA
complementary to these sequences on opposite strands of the target sequence is used as
primers.
o. DNA that has a known size or allele set. This DNA is often compared to the sample
DNA being tested.
2. List the materials required for PCR.
1.
2.
3.
4.
5.
38
3. Put the steps of PCR in the correct order.
-heat mixture to allow DNA polymerase to synthesize new DNA complementary to
the target DNA
-heat mixture to unzip the target DNA
-cool mixture to allow the primers to bond to the flanking DNA
4. Who invented PCR? When?
5. From one cell, one billion copies of the target sequence can be copied using PCR in
approximately
hours.
6. The entire genome is copied at one time using PCR.
a. True
b. False
7. PCR has a disadvantage. It can be too
lead to
which may
.
8. Sequences on either side of the target sequence are called
sequences.
9. Small DNA molecules complementary to flanking sequences that are synthesized in
the lab are called…
10. The enzyme which bonds the free DNA nucleotides to the complementary target
DNA during PCR is called…
39
11. What is the genotype of person #5 for the vWA STR?
12. How are STR fragments “seen” in a capillary electrophoresis apparatus?
13. Describe the advantages of a capillary electrophoresis system.
40
14. Describe how researchers can ‘ignore’ the portion of DNA not to be tested and only
‘see’ the areas of the DNA to be tested: RFLP and PCR/STRs.
15. Compare the procedures used to test and the characteristics of
1) RFLP LTRs and PCR/ STRs.
16. Describe, IN DETAIL, why the primers used during PCR are also used as probes
when detecting STR alleles.
17. Describe how one can simultaneously amplify many STRs in one tube.
41
18. Describe, in detail, how the ladder DNA is used to determine the number of repeats in
the sample DNA being tested.
42