Transcript DNA Analysis Part II: RFLPs, STRs, and Sequencing


How does the graph represent a gel?
Each group filled in a ‘band’ that represents where
different – sized DNA fragments would have migrated on a gel,
with smaller fragments migrating farther.

Why is each group’s ‘lane’ different?
Each group had a unique DNA sequence, and so that
fragments produced by cutting with the restriction
enzyme differ.

Is it possible for two different people to produce the
same RFLP pattern?
Yes, but it is very unlikely, especially if multiple different
restriction enzymes are used. Moreover, the odds can be
estimated.
A woman is violently
killed, and her hands
have defensive injuries
indicating that she
fought her attacker.
Investigators find skin cells
under her nails.
They extract the DNA
from these cells, amplify
it, and run an RFLP
analysis.
Do any of the suspects
have matching DNA?
Different RFLPs are
referred to as alleles.
To analyze RFLPs for
paternity, remember:
any alleles in a child that
didn’t come from mother
must come from father.
Who is the father?
 First match child to
mom
 Then, match
remaining alleles to
father
Why can’t #1 or #3 be the
father?
Neither has all the ‘missing’
alleles.
Is the real father the one
who shares the most alleles
with the child?
No. Its possible to have a lot of
alleles in common and not be
the dad.
Can you determine
paternity without having
the mother’s DNA?
Not using this method, and not
as accurately.
Is Darth Vader
really Luke’s
father?
Justify your answer by
matching the alleles.
Yes – he is the father.
Every allele in Luke &
Leia match an allele
found in Anakin’s or
Padme’s results.
But there’s still a
problem … see it?
Luke and Leia aren’t
identical twins!
Dad #4
All of the child’s
alleles match up
with either mom
or dad #4.
Is there any other time when you might
need to compare the DNA of family
members to solve a crime?
Missing persons cases
Watch me!

STR stands for short tandem repeat

These are places (loci) in DNA that contain a variable
number of a short, repeated sequence of
nucleotides.
E.g. AGCAGCAGCAGCAGCAGCAGC (7 repeats)
or
AGCAGCAGCAGCAGC (5 repeats)

The number of repeats are person has is their allele
for that STR loci.

Each person has two versions of every chromosome,
so they have 2 alleles. (E.g. 6 , 10 for a particular
STR)
1.
DNA is collected and extracted.
2.
Regions containing each STR loci are amplified and
separated according to size using automated
technologies.
Why automated technology?
Because the difference in size between different alleles can
be miniscule – far too small to see in a gel done by hand.
3.
Evidence can be matched to a suspect and/or
entered into a DNA database (CODIS). In the US,
investigator routinely analyze 13 STR loci.
4.
Frequencies of different alleles are known, so that
probability of random matches can be calculated.

What do the
numbers mean?
That is the number of
repeats for that loci

Why are their two
numbers per loci?
Each person has two
alleles per loci

Why are some
alleles not whole
numbers?
Some repeats may
not be completed
(e.g. ACGACGA)

Which suspect
matches the
evidence?
Suspect B

How could we
calculate the
probability that the
suspect matched by
chance?
If we knew the
frequency of each
allele in the general
population, we could
multiply all the
probabilities.
Calculate the
frequency of this
combination of
STR genotypes.
.000000006%
Or
About 6 in 10
billion
Is it likely that someone else in the
world has this same genotype?
Yes! The world has 7 billion
people.
What does this mean for matching
a suspect to a crime scene?
What about for matching a
sample to a DNA data base?
Who is the
baby
daddy?
Richard
How do you
know?
- Every allele
in the child
came from
mother or
Richard
How can
you exclude
the others?
STR Loci
Mother
Child
Stephen
Richard Charles
D35S1358
15, 16
15, 17
16, 17
17, 17
15, 15
vWA
16, 19
15, 16
16, 20
15, 19
15, 16
FGA
21, 21
21, 23
22, 26
23, 27
23, 26
D8S1179
12, 15
13, 15
15 , 15
12 , 13
11, 13
D21S11
30, 30.2 30.2, 30.2
30, 30.2
28, 30.2
30, 30.2
D18S51
18, 18
12, 18
14, 18
12, 12
15, 18
D5S818
12 , 13
13, 13
10, 13
9, 13
10, 12
D13S317
11 , 12
12 , 12
12 , 13
11 , 12
11, 12
D7S820
10 , 11
10, 11
11, 12
11, 13
10, 13
CSF1PO
8, 8
8, 11
9, 9
9, 11
9, 11
TPOX
7,8
8, 8
8, 10
8, 10
7, 10
THO1
6, 9.3
9.3, 9.3
6, 6
6, 9.3
6, 8
D16S539
11, 13
9, 13
11, 13
9, 12
11, 12

DNA is amplified using a small fraction of special
nucleotides that will end the formation of a chain early.
› Since the inclusion of these special nucleotides is random
and since the process is repeated MANY MANY MANY times,
you eventually wind up with a set of different strands, each
one base pair different in length, each ending in a special
nucleotide.

These special, ending nucleotides are labeled in some
way (i.e. radioactively, with dye, etc.) so that a
machine can tell the difference between A, C, G, and T

These fragments are then sorted by size, and the special
nucleotides are ‘read’ by the machine to form the
sequence.
Watch me!
What were our objectives, and what did
we learn?
 What was our learner profile trait and
how did we demonstrate it?
 How does what we did today tie to our
unit question?
