An Introduction to Bioinformatics Algorithms www.bioalgorithms.

info

DNA Mapping and Brute

Force Algorithms!
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Outline!
•  Restriction Enzymes
•  Gel Electrophoresis
•  Partial Digest Problem
•  Brute Force Algorithm for Partial Digest
Problem
•  Branch and Bound Algorithm for Partial
Digest Problem
•  Double Digest Problem
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Molecular Scissors!

Molecular Cell Biology, 4th edition

An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Discovering Restriction Enzymes!

•  HindII - first restriction enzyme – was

discovered accidentally in 1970 while
studying how the bacterium Haemophilus
influenzae takes up DNA from the virus
•  Recognizes and cuts DNA at sequences:
•  GTGCAC
•  GTTAAC
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Discovering Restriction Enzymes!

My father has discovered a servant
who serves as a pair of scissors. If
a foreign king invades a bacterium,
this servant can cut him in small
fragments, but he does not do any
harm to his own king. Clever
people use the servant with the
Werner Arber Daniel Nathans Hamilton Smith scissors to find out the secrets of
the kings. For this reason my father
Werner Arber – discovered restriction received the Nobel Prize for the
enzymes discovery of the servant with the
Daniel Nathans - pioneered the application
scissors".
of restriction for the
construction of genetic
maps Daniel Nathans daughter
Hamilton Smith - showed that restriction (from Nobel lecture)
enzyme cuts DNA in the
middle of a specific sequence
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Recognition Sites of Restriction Enzymes!

Molecular Cell Biology, 4th edition

An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Uses of Restriction Enzymes!

•  Recombinant DNA technology

•  Cloning

•  cDNA/genomic library construction

•  DNA mapping
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Restriction Maps!
• A map showing positions
of restriction sites in a
DNA sequence
• If DNA sequence is
known then construction
of restriction map is a
trivial exercise
• In early days of
molecular biology DNA
sequences were often
unknown
• Biologists had to solve
the problem of
constructing restriction
maps without knowing
DNA sequences
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Full Restriction Digest!

• Cutting DNA at each restriction site creates

multiple restriction fragments:

• Is it possible to reconstruct the order of

the fragments from the sizes of the fragments
{3,5,5,9} ?
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Full Restriction Digest: Multiple Solutions !

• Alternative ordering of restriction fragments:

vs
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Measuring Length of Restriction Fragments!

•  Restriction enzymes break DNA into restriction

fragments.

•  Gel electrophoresis is a process for separating

DNA by size and measuring sizes of restriction
fragments

•  Can separate DNA fragments that differ in length in

only 1 nucleotide for fragments up to 500
nucleotides long
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Gel Electrophoresis!
•  DNA fragments are injected into a gel
positioned in an electric field
•  DNA are negatively charged near neutral pH
•  The ribose phosphate backbone of each

nucleotide is acidic; DNA has an overall

negative charge
•  DNA molecules move towards the positive
electrode
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Gel Electrophoresis (cont d)!

•  DNA fragments of different lengths are

separated according to size
•  Smaller molecules move through the gel

matrix more readily than larger molecules

•  The gel matrix restricts random diffusion so
molecules of different lengths separate into
different bands
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Gel Electrophoresis: Example!

Direction of DNA
movement

Smaller fragments
travel farther

Molecular Cell Biology, 4th edition

An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Detecting DNA: Autoradiography!

•  One way to visualize separated DNA
bands on a gel is autoradiography:
•  The DNA is radioactively labeled
•  The gel is laid against a sheet of
photographic film in the dark, exposing
the film at the positions where the DNA is
present.
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Detecting DNA: Fluorescence!

•  Another way to visualize DNA bands in gel
is fluorescence:
• The gel is incubated with a solution
containing the fluorescent dye ethidium
• Ethidium binds to the DNA
• The DNA lights up when the gel is
exposed to ultraviolet light.
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Restriction Digest!

•  The sample of DNA is exposed to the
restriction enzyme for only a limited amount of
time to prevent it from being cut at all restriction
sites
•  This experiment generates the set of all
possible restriction fragments between every
two (not necessarily consecutive) cuts
•  This set of fragment sizes is used to determine
the positions of the restriction sites in the DNA
sequence
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Digest Example!

•  Partial Digest results in the following 10 restriction
fragments:
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Multiset of Restriction Fragments!

•  We assume
that multiplicity
of a fragment
can be
detected, i.e.,
the number of
restriction
fragments of
the same
length can be
determined
(e.g., by
observing twice
as much
fluorescence
intensity for a
double Multiset: {3, 5, 5, 8, 9, 14, 14, 17, 19, 22}
fragment than
for a single
fragment)
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Digest Fundamentals!

X: the set of n integers representing the

location of all cuts in the restriction map,
including the start and end
n: the total number of cuts
DX: the multiset of integers representing
lengths of each of the fragments
produced from a partial digest
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

One More Partial Digest Example!

X! 0! 2! 4! 7! 10!
0! !
2! 4! 7! 10!
2! ! !
2! 5! 8!
4! ! ! !
3! 6!
7! ! ! ! !
3!
10!
Representation of DX ! = {2,! 2, !3, 3,! 4, 5,
! 6, 7, 8, 10} as a two
dimensional table, with elements of
X = {0, 2, 4, 7, 10}
along both the top and left side. The elements at (i, j) in the table
is xj – xi for 1 ≤ i < j ≤ n.
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Digest Problem: Formulation!

Goal: Given all pairwise distances between

points on a line, reconstruct the positions of
those points

•  Input: The multiset of pairwise distances L,

containing n(n-1)/2 integers
•  Output: A set X, of n integers, such that
DX = L
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Digest: Multiple Solutions!

•  It is not always possible to uniquely reconstruct a set X based
only on DX.
•  For example, the set
X = {0, 2, 5}
and
(X + 10) = {10, 12, 15}
both produce DX={2, 3, 5} as their partial digest set.
•  The sets {0,1,2,5,7,9,12} and {0,1,5,7,8,10,12} present a less
trivial example of non-uniqueness. They both digest into:
{1, 1, 2, 2, 2, 3, 3, 4, 4, 5, 5, 5, 6, 7, 7, 7, 8, 9, 10, 11, 12}
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Homometric Sets!
0 1 2 5 7 9 12 0 1 5 7 8 10 12
! !

0 1 2 5 7 9 12 0 1 5 7 8 10 12
! !

1 1 4 6 8 11 1 4 6 7 9 11
! ! ! !

2 3 5 7 10 5 2 3 5 7
! ! ! ! ! !

5 2 4 7 7 1 3 5
! ! ! ! ! ! ! !

7 2 5 8 2 4
! ! ! ! ! ! ! ! ! !

9 3 10 2
! ! ! ! ! ! ! ! ! ! ! !

12 12
! ! ! ! ! ! ! ! ! ! ! ! ! !
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Brute Force Algorithms!

•  Also known as exhaustive search
algorithms; examine every possible variant
to find a solution

•  Efficient in rare cases; usually impractical

An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Partial Digest: Brute Force!

1.  Find the restriction fragment of maximum length
M. M is the length of the DNA sequence.
2.  For every possible set
X={0, x2, … ,xn-1, M}
compute the corresponding DX
5.  If DX is equal to the experimental partial digest
L, then X is the correct restriction map
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

BruteForcePDP!
1.  BruteForcePDP(L, n):!
2.  M <- maximum element in L"
3.  for every set of n – 2 integers 0 < x2 < … xn-1 < M"
4.  X <- {0,x2,…,xn-1,M}!
5.  Form DX from X"
6.  if DX = L"
7.  return X"
8.  output no solution !
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Efficiency of BruteForcePDP!
•  BruteForcePDP takes O(M n-2) time since it
must examine all possible sets of positions.

•  One way to improve the algorithm is to limit

the values of xi to only those values which
occur in L.
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

AnotherBruteForcePDP!
1.  AnotherBruteForcePDP(L, n)!
2.  M <- maximum element in L"
3.  for every set of n – 2 integers 0 < x2 < … xn-1 < M"
4.  X <- { 0,x2,…,xn-1,M }!
5.  Form DX from X"
6.  if DX = L"
7.  return X"
8.  output no solution !
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

AnotherBruteForcePDP!
1.  AnotherBruteForcePDP(L, n)!
2.  M <- maximum element in L"
3.  for every set of n – 2 integers 0 < x2 < … xn-1 < M from L"
4.  X <- { 0,x2,…,xn-1,M }!
5.  Form DX from X"
6.  if DX = L"
7.  return X"
8.  output no solution !
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Efficiency of AnotherBruteForcePDP!

•  It s more efficient, but still slow

•  If L = {2, 998, 1000} (n = 3, M = 1000),
BruteForcePDP will be extremely slow, but
AnotherBruteForcePDP will be quite fast
•  Fewer sets are examined, but runtime is still
exponential: O(n2n-4)
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Branch and Bound Algorithm for PDP!

1.  Begin with X = {0}

2.  Remove the largest element in L and place
it in X
3.  See if the element fits on the right or left
side of the restriction map
4.  When it fits, find the other lengths it creates
and remove those from L
5.  Go back to step 1 until L is empty
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Branch and Bound Algorithm for PDP!

1.  Begin with X = {0}

2.  Remove the largest element in L and place
it in X
3.  See if the element fits on the right or left
side of the restriction map
4.  When it fits, find the other lengths it creates
and remove those from L
5.  Go back to step 1 until L is empty
WRONG ALGORITHM
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Defining D(y, X) !

•  Before describing PartialDigest, first define

D(y, X)
as the multiset of all distances between point y
and all other points in the set X
D(y, X) = {|y – x1|, |y – x2|, …, |y – xn|}!
for X = {x1, x2, …, xn}!
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

PartialDigest Algorithm!

PartialDigest(L):!
width <- Maximum element in L"
DELETE(width, L)!
X <- {0, width}!
! PLACE(L, X)!
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

PartialDigest Algorithm (cont d)!

1.  PLACE(L, X)!

2.  if L is empty!
3.  output X"
4.  return!
5.  y <- maximum element in L"
6.  Delete(y,L)"
7.  if D(y, X ) Í L"
8.  Add y to X and remove lengths D(y, X) from L"
9.  PLACE(L,X )!
10.  Remove y from X and add lengths D(y, X) to L"
11.  if D(width-y, X ) Í L"
12.  Add width-y to X and remove lengths D(width-y, X) from L"
13.  PLACE(L,X )!
14.  Remove width-y from X and add lengths D(width-y, X ) to L"
15.  return!
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0 }!
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0 }!
"
Remove 10 from L and insert it into X. We know this must be
the length of the DNA sequence because it is the largest
fragment.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 10 }!
"
"
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 10 }!
"
Take 8 from L and make y = 2 or 8. But since the two cases
are symmetric, we can assume y = 2.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 10 }!
"
We find that the distances from y=2 to other elements in X are
D(y, X) = {8, 2}, so we remove {8, 2} from L and add 2 to X.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 10 }!
"
"
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 10 }!
"
Take 7 from L and make y = 7 or y = 10 – 7 = 3. We will
explore y = 7 first, so D(y, X ) = {7, 5, 3}.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 10 }!
"

For y = 7 first, D(y, X ) = {7, 5, 3}. Therefore we

remove {7, 5 ,3} from L and add 7 to X.

D(y, X) = {7, 5, 3} = {½7 – 0½, ½7 – 2½, ½7 – 10½}!

 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 7, 10 }!
"
!
!
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 7, 10 }!

This time make y = 4. D(y, X) = {4, 2, 3 ,6}, which is a

subset of L so we will explore this branch. We remove
{4, 2, 3 ,6} from L and add 4 to X.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 4, 7, 10 }!
"
"
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 4, 7, 10 }!
"
L is now empty, so we have a solution, which is X.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 7, 10 }!
"
To find other solutions, we backtrack.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 10 }!
"
More backtrack.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 2, 10 }!
"
This time we will explore y = 3. D(y, X) = {3, 1, 7}, which is
not a subset of L, so we won t explore this branch.
 An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

An Example!
L = { 2, 2, 3, 3, 4, 5, 6, 7, 8, 10 }!
X = { 0, 10 }!
"
We backtracked back to the root. Therefore we have found
all the solutions.
An Introduction to Bioinformatics Algorithms www.bioalgorithms.info

Analyzing PartialDigest Algorithm!

•  Still exponential in worst case, but is very fast

on average
•  Informally, let T(n) be time PartialDigest takes
to place n cuts
•  No branching case: T(n) < T(n-1) + O(n)"

•  Quadratic

•  Branching case: T(n) < 2T(n-1) + O(n)"

•  Exponential