Transcript Physical Mapping I

Physical Mapping I
CIS 667 February 26, 2004
Physical Mapping
• A physical map of a piece of DNA tells us
the location of certain markers
 A marker is a short sequence
 Given a sequence and a chromosome - try to
find the place of the sequence
G
A
B
G
C
C
H
D
Physical Mapping
• Generally used to resolve regions much
larger than 1 Mb (e.g. whole
chromosomes)
• Map is created by fragmenting the DNA
molecule using restriction enzymes and
then looking for overlaps
 The pieces are too big to sequence, so this is
not the same problem as fragment assembly!
An Example - Cystic Fibrosis
• Cystic fibrosis is a fatal disease
 1 in 25 Caucasians carries a faulty cystic
fibrosis gene
 Children who inherit faulty genes from both
parents become sick
 Best hope for a cure starts with finding the
responsible gene
 In the mid 80s nothing was known about the
CF gene so a search was started for it
An Example - Genetic Mapping
• Restriction fragment length polymorphism
(RFLP) was used to construct a map of
the human genome with one marker every
10 million nucleotides
 RFLP based on variability of certain
nucleotides between different people
 These cause restriction fragments of different
lengths to be produced
An Example - Genetic Mapping
• A statistical study of 21 families over three
generations narrowed the search led to an
area of length 1 million bp on chromosome
7 between 2 markers
 The presence of two phenotypes together
more often leads to the conclusion that the
genes are physically close due to the way
crossover works to reshuffle genomic material
An Example - Physical Mapping
• Now physical mapping was needed to
more precisely locate the CF gene
• The DNA molecule was broken into pieces
50 Kb long
 Now the correct ordering of the pieces had to
be obtained
 They are cloned to obtain a large number of
copies in a clone library
 Now the clones must be ordered
An Example - Physical Mapping
• The idea is to describe each clone using a
fingerprint to describe the clones
 Can be thought of as “key words” for the
clones
 Overlapping clones should have similar
fingerprints
X Y Z Q
An Example - Physical Mapping
• Fingerprints can be
 Sizes of restriction fragments of clones
 List of probes hybridizing to the clones
• The CF gene was close to RFLP DS78
marker
 A probe for this RFLP was used to find a
clone containing it
 The clone was sequenced at the end and a new
probe designed to move closer to the gene (chromosome walking)
Physical Mapping
• Two generic ways of obtaining fingerprints
are
 Restriction site analysis
 Hybridization
• Restriction site analysis locates the
restriction sites of an enzyme on the target
DNA
 Generally applicable with smaller DNAs
 Viral and mitochondrial DNA
Physical Mapping
• In hybridization mapping we check
whether certain small sequences bind to
fragments
 More widely used nowadays, especially for
large scale physical mapping
• Due to lack of information and errors, we
may not be able to produce a single
contiguous physical map
 May have lack of coverage or chimeric clones
Restriction Site Mapping
• There are two techniques for measuring
the length of fragments between restriction
sites
 Apply two different restriction enzymes to the
target DNA
 Each enzyme cuts at a different location
 Apply enzyme A, apply enzyme B, and also apply
both A and B together
 Now we have a three sets of fragments of various
lengths
Restriction Site Mapping
• The discovery of the original ordering of
these fragments is called the double digest
problem
Partial Digest Problem
• A variant of the double digest approach is
partial digest
 Use just one enzyme, but for varying amounts
of time
 Fragments of different lengths will be
produced
Restriction Site Mapping
• Problems
 Uncertain lengths
 Gel electrophoresis gives error up to 5%
 Fragments may be too short to measure
 May lose fragments
Hybridization Mapping
• Done using markers called STS
(Sequenced Tagged Sites)
 Uses PCR techniques to identify unique
sequences
 Verify whether the clone allows some probes
to bind (hybridize)
 See if the clones have overlapping sets of
binding probes
Hybridization Mapping
• Problems
 False negative
 False positive
 Chimeric clones
 Deletion
 Repeats (not for STS)