Computat onal Biology

Lecture 2

Saad Mneimneh
http://engr.smu.edu/~saad/
 Genetic mapping
• Single chromosome with n genes

• Single recombination point that occurs uniformly at random

• Probability of recombination between two genes at distance d is p=d/(n+1)

• Estimate p (and therefore d) by observing the frequency of different

phenotypes

d
• Problems
– Too many chromosomes
– Not all genes have phenotypes that can be observed
– We usually don’t know what we are looking for
Saad Mneimneh
http://engr.smu.edu/~saad/
RFLP: Restriction Fragment Length
Polymorphism
• A restriction enzyme cuts the DNA molecules at every occurrence of
a particular sequence, called restriction site.

• For example, HindII enzyme cuts at GTGCAC or GTTAAC

• If we apply a restriction enzyme on DNA, it is cut at every

occurrence of the restriction site into a million restriction fragments,
each a few thousands nucleotides long.

• Any mutation of a single nucleotide may destroy or create the site

(CTGCAC or CTTAAC for HindII) and alter the length of the
corresponding fragment.

• RFLP analysis is the detection of the change in the length of the

restriction fragments.

Saad Mneimneh
http://engr.smu.edu/~saad/
 Gel-Electrophoresis
• DNA is cut into fragments using an enzyme
• The cut DNA is put on a Gel material
• An electric current is applied on the Gel
• DNA is negatively charge
• DNA fragments will start moving towards the
positively charged side
• Smaller fragments move faster
• After some time, we have a separation of the
different fragment lengths

Saad Mneimneh
http://engr.smu.edu/~saad/
 DNA Sample
• Some cells are obtained

• The cells are immersed in

a nutritious solution on a
plate and left to grow and
multiply

• The cells are gathered

and frozen for future use

• Liquidized DNA is
obtained from these cells

Saad Mneimneh
http://engr.smu.edu/~saad/
 Restriction Enzyme

• A restriction enzyme
is used to cut the
DNA into fragments

• Hind III restriction site

is A AGCTT

Saad Mneimneh
http://engr.smu.edu/~saad/
 Apply Enzyme

• DNA sample and

Hind III are put
together in a tube

• The tube is shaken by

rotation for DNA and
Hind III to mix

Saad Mneimneh
http://engr.smu.edu/~saad/
 Water Bath
• The tube is put on a
plate floating on water
at 37oC

• It is left for 30 minutes

• This is needed for the

Hind III reaction to
take place

Saad Mneimneh
http://engr.smu.edu/~saad/
 Preparing the Gel

• In the meantime, we
prepare the Gel

• Agarose powder is
the basic substance
for making the Gel

Saad Mneimneh
http://engr.smu.edu/~saad/
 Preparing the Gel

• The powder is mixed

with water in a
container

Saad Mneimneh
http://engr.smu.edu/~saad/
 Preparing the Gel

• The container is
heated (in a
microwave if you
want) until the powder
completely dissolves
in the water

• The solution becomes

clear

Saad Mneimneh
http://engr.smu.edu/~saad/
 Preparing the Gel
• The liquid Gel is poured into
the inner box comb like piece

• A comb like piece is put at the

edge of the inner box

• The liquid Gel is left to cool

and solidify (you can use a
fridge)

• When the Gel solidifies, the

comb will create wells for the
DNA samples to be put inner box
H shaped
container

Saad Mneimneh
http://engr.smu.edu/~saad/
 Gel Ready
• Gel ready
• Fill the H shaped
container with water
• Remove comb
well

Saad Mneimneh
http://engr.smu.edu/~saad/
 Putting DNA on the Gel
original DNA cut by
uncut DNA Hind III
• DNA samples mixed with
colored solution and UV
reactive solution

• DNA samples inserted

into wells

• A sample DNA containing

only specific fragments
(called ladder) can be ladder 1 ladder 2
used for comparison

Saad Mneimneh
http://engr.smu.edu/~saad/
 Run the Gel

• Apply electric current

• DNA is negatively
charged

• Fragments will migrate

toward the positive
charge

• Small fragments move

faster

Saad Mneimneh
http://engr.smu.edu/~saad/
 DNA Fragments Move

• The colored solution

provides an indication
start
to how much the DNA
has traveled on the
Gel

Saad Mneimneh
http://engr.smu.edu/~saad/
 Viewing

• Gel can be viewed

under UV light

Saad Mneimneh
http://engr.smu.edu/~saad/
 Viewing
• Original uncut DNA sample
makes a sharp band at the
beginning (one big fragment)

• DNA sample cut with Hind III

makes s smear (lots of
fragments of all sizes)

• Ladders are used for

comparison (they contain
specific fragments)

• We could run it for a longer

time to achieve better
separation

Saad Mneimneh
http://engr.smu.edu/~saad/
 Hybridization
• In a hybridization experiment, we try to
verify whether a specific sequence known
as probe binds (or hybridizes) with a DNA
fragment.

• If the binding occurs, this means that the

DNA fragment contains the sequence
complementary to the probe sequence (or
parts of it).

Saad Mneimneh
http://engr.smu.edu/~saad/
 RFLP Markers
• We apply a number of probes in turn on the gel
• Each probe is mixed with a radioactive material
• Each probe hybridizes with a portion of the original DNA
• After cutting, the probe will hybridize with the fragments belonging to that
portion
• These fragment can now be observed due to the radioactive material
• RFLP marker is defined by a probe and the set of lengths (unordered) of
fragments that hybridize with the probe.
• Use analysis of recombination to order RFLP markers on the chromosome

RFLP marker
probe 1 probe 2

restriction enzyme cuts

restriction
site
restriction
fragment
Saad Mneimneh
http://engr.smu.edu/~saad/
 Illustration
probe cut DNA

smear
fragments contained
in the probe

Gel

Saad Mneimneh
http://engr.smu.edu/~saad/
 First RFLP map in 1987
• Donis-Keller et al. constructed the first RFLP map of the
human genome, positioning one RFLP marker per
approximately 10 million nucleotides.

• RFLP markers (probes) need to be long enough to span

the whole DNA.

• 393 random probes where used to study RFLP in 21

families over 3 generations.

• Computational analysis of recombination lead to the

ordering of RFLP markers on the chromosome.

Saad Mneimneh
http://engr.smu.edu/~saad/
 RFLP and Gene Finding
• Using the ordering of RFLP markers on a chromosome,
we can approximately determine the location of a gene.

– How?
– Find the difference between the RFLP markers of family
members with the disease and family members not having the
disease.
– It is likely that the RFLP marker that consistently differ is on the
gene responsible for the disease, since family members have
more or less the same genetic characteristics.
– But we still don’t know where and what the exact gene is.

Saad Mneimneh
http://engr.smu.edu/~saad/
 Physical Mapping
• Genetic mapping and RFLP
– (1) do not tell the actual distance in base pairs
– (2) if genes (or markers) are very close, one cannot
resolve their order, because the observed
recombination frequencies will be zero.

• Physical mapping reflect actual distances

– Hybridization Mapping
– Restriction Mapping

Saad Mneimneh
http://engr.smu.edu/~saad/
 Hybridization Mapping
• Break several copies of DNA into fragments (using different restriction
enzymes).

• Obtain many copies of each fragment (cloning, incorporating a fragment into

a replicating host), forming a clone library.

• Clones may overlap (cutting DNA with distinct enzymes), and we want them
to (we will see why).

• Fingerprinting the clones: Now use DNA probes, and for every clone
determine the list of probes that hybridize with the clone

• When two clones have substantial overlap, their fingerprints will be similar.

• Reconstruct the relative order of the clones using the overlap information
(this order is unknown in RFLP)

Saad Mneimneh
http://engr.smu.edu/~saad/
 Hybridization Mapping
• For n clones, and m probes, the hybridization
data consists of an n x m matrix D, such that
dij=1 if clone Ci contains probe pj.

• Let S be a string over the alphabet of probes

p1…pm. S covers a clone C if there exists a
substring of S containing exactly the same set of
probes as C (order and multiplicity are ignored)

• A simple approximation of physical mapping is

the Shortest Covering String.

Saad Mneimneh
http://engr.smu.edu/~saad/
 Illustration

No overlap
No information

Covering String:

Saad Mneimneh
http://engr.smu.edu/~saad/
 Covering string

The clone is covered by the string

String of probes

This clone hybridizes with 4 probes

Saad Mneimneh
http://engr.smu.edu/~saad/
 Shortest Covering String
probes
A B C D E F G
1 1 1 CAEBGCFDAGEBAGD
2 1 1 1 1 1
3 1 1 1 1 1 1 2
4 1 1 1 1 1 3
4
clones 5 1 1 1 1 5
6 1 1 1 1 6
7 1 1 1 1 1 7
8
8 1 1 1 1 9
9 1
A covering string: S= AC ABEG BCDEFG BCDFG CDFG ADFG ABDEG ABDG D

A shortest covering string (max overlap): S= C A E B G C F D A G E B A G D

Shortest Covering String: NP-hard Problem in general. If probes are unique, a

polynomial algorithm exists.
Saad Mneimneh
http://engr.smu.edu/~saad/
 Unique/Non-Unique Probes

• Non-unique probes: probes are short random sequences

that can occur many times in the DNA. Therefore, a
probe can hybridize with distant clones.

• Unique probes: probes are sufficiently long and are

unlikely to occur twice in the DNA. Therefore, a probe
will hybridize with close clones.

• Advantages of non-unique probes: probe generation is

cheap and straight-forward.

Saad Mneimneh
http://engr.smu.edu/~saad/
 Restriction Mapping
• Before using the list of probes in a clone as a
fingerprint, biologists used the order of
restriction fragments in a clone.

• Restriction map as Fingerprinting: If two clones

share several consecutive fragments, they are
likely to overlap.

• Restriction map of a clone: an ordered list of its

restriction fragments (Hard Problem).

Saad Mneimneh
http://engr.smu.edu/~saad/
 Double Digest
• Cut the DNA fragment with enzyme A, then enzyme B,
then both

• Obtain a multiset of lengths in each case (using Gel

electrophoresis)

• Using this information, construct an order of the lengths

• A: {2,2,3} 2 3 2
• B: {3,4} 3 4
• A+B: {1,2,2,2} 2 1 2 2

Saad Mneimneh
http://engr.smu.edu/~saad/
 Partial Digestion
• Instead of obtaining lengths of restriction fragments, the DNA is digested in
such a way that fragments are formed by every two cuts and the lengths of
all fragments are obtained.

• The problem often might be formulated as recovering positions of points on

a line when only some pairwise distances between points are known.
(why?)

• Many mapping techniques lead to the following problem: X is a set of points,

∆X is the multiset of all pairwise distances between points in X: ∆X={|x1-x2| :
x1 , x2 ∈ X}, E ⊆ X is given. Reconstruct X from knowing E alone.

• Partial Digest Problem. Given ∆X, reconstruct X (E=∆X). Also known as the
turnpike problem in computer science, construct the geography of the
highway from knowing the distance between every two exits.

• No polynomial time algorithm for this problem is yet known, but in practice,
efficient algorithms exist.

Saad Mneimneh
http://engr.smu.edu/~saad/