The document discusses the necessity of physical mapping in genome projects, highlighting the limitations of genetic maps that are often insufficient for sequencing direction due to resolution issues and inaccuracies. It describes physical maps, which utilize DNA sequence landmarks and are created using techniques like restriction mapping, FISH, and STS mapping. Additionally, it explains the methods and challenges associated with restriction mapping and pulsed field gel electrophoresis (PFGE) for handling large DNA fragments.

1. Physical mapping

2. Steps to map the GEnome
Markers to
generate
highly
saturated map
Physical map
Sequencing
for the whole
genome

3. Steps to map the GEnome
Markers to
generate
highly
saturated map
Physical map
Sequencing
for the whole
genome

4. Why we need to make physical mapping?

5. Why we need physical mapping?
A map generated by
genetic techniques is
rarely sufficient for
directing the sequencing
phase of a genome project
The resolution of a
genetic map depends
on the number of c.o.
that have been scored

6. Why we need physical mapping?
The resolution of a genetic map depends
on the number of c.o. that have been
scored:
• it is not possible in eukaryotes to
obtain large number of progeny, so
relatively few meiosis can be studied.
• The resolving power of linkage analysis
is restricted (markers that are several
tens of kb apart may appear at the
same position).

7. Why we need physical mapping?
A map generated by
genetic techniques is
rarely sufficient for
directing the sequencing
phase of a genome project
The resolution of a
genetic map depends
on the number of c.o.
that have been scored
Genetic maps
have limited
accuracy

8. Why we need physical mapping?
Genetic maps have limited accuracy:
• Genetic maps assume that c.o. occur at
random along chromosomes.
• The presence of recombination
hotspots means that c.o. are more
likely to occur at some points rather
than at others.

9. Why we need physical mapping?
A map generated by
genetic techniques is
rarely sufficient for
directing the sequencing
phase of a genome project
The resolution of a
genetic map depends
on the number of c.o.
that have been scored
Genetic maps
have limited
accuracy

10. For most eukaryotes, a genetic map must be checked and
supplemented by physical mapping techniques before large-scale
DNA sequencing begins

11. Genetic maps
• Abstract maps that place the
relative location of genes or
markers on chromosomes
based on recombination
frequency.
• Distances between markers is
measured in centimorgan.
• Different markers could be used
to generate genetic maps:
1. Morphological
2. Biochemical
3. molecular.
Physical maps
• Use landmarks within DNA sequences
ranging from restriction sites to the
actual DNA sequence.
• Distances between “landmarks” are
measured in base-pairs ( bp).
• Knowledge of DNA sequence is not
necessary
• Three main techniques for generating
physical maps :
1. Restriction mapping
2. FISH
3. STS mapping .

12. Most important physical mapping
techniques
Restriction
mapping.
Fluorescent
in situ
hybridization
(FISH).
Sequence
tagged sites
(STS)
mapping.

13. Most important physical mapping
techniques
Restriction
mapping.
Fluorescent
in situ
hybridization
(FISH).
Sequence
tagged sites
(STS)
mapping.

14. Restriction mapping
• The first physical maps
• Locates the relative positions on a DNA
molecule of the recognition sequences for
restriction endonucleases
• Based on distances between restriction
sites
• Overlap between smaller segments can be
used to assemble them into a contig
(Continuous segment of the genome).

16. The scale of restriction mapping is
limited by the sizes of the restriction
fragments
• As the number of cut sites increases
• the numbers of single, double and partial restriction products
increases.
• Restriction mapping is more applicable to small rather than large
molecules
• Computer analysis can be used but problems still eventually arise.

17. If DNA molecule
>50 kb
• possible to construct an
unambiguous restriction map for
a selection of enzymes with 6 bp
recognition sequences (this
could cover a few viral and
organelle genomes).
• a detailed restriction map can
then be built up from the cloned
fragments(>50) as a preliminary
to sequencing the cloned region
<50
• there is a possibility of using
restriction analysis for mapping
genomes larger than 50kb by
choosing enzymes expected to
have infrequent cut sites "rare
cutters" in the target DNA
molecule.

18. Rare cutters fall into 2
categories
Enzymes with 7 or 8 nucleotide
recognition sequences
• Sap I (5'- GCTCTTC-3’)
• cuts every 4^7 = 16 384 bp
• Sgf I (5' - GCGA TCGC-3') cuts
every 4^8 = 65 536 bp.
Enzymes whose recognition sequences
contain motifs that are rare in the target
DNA
• SmaI (5'-CCCGGG-3‘)
• Bss HII(5'-GCGCGC-3')

19. To separate large DNA fragments, it
is necessary to replace linear
electric field with a more complex
field
In an electrophoretic gel, the resolution decreases as the molecules get
longer.
Thus, molecules > 50 kb in length run as a single slowly migrating band
in a standard agarose gel

20. Pulsed Field Gel Electrophoresis (PFGE)
• PFGE resolves DNA molecules of 100 - 1 000
kb
• changing the direction of the electric field in a
way that causes large DNA fragments to re-
align more slowly with the new field direction
than do smaller molecules

21. Basic types of PFGE systems
Field inversion PFGE (FIGE)
• Using a standard gel box
apparatus
• Works by periodically reversing
the direction of the electric field
Contour clamped Homologous electric
field (CHEF)
• Using hexagonal box
• Works by multiple electric fields

22. Standard biochemical extraction procedures

23. Standard biochemical extraction procedures

24. PFGE DNA samples are prepared using
immobilized cells in agar blocks
• very large DNA fragments are randomly
sheared by physical forces
• soaked in lysis buffer containing detergent and
proteinase K. This method gently purifies the
cellular DNA without subjecting it to the
shearing forces produced by pipetting.
• Following cell lysis, a protease inhibitor is
soaked into the agar block to inactivate the
proteinase K and the DNA is then digested with
RE prior to placing the agar block into the well
of the PFGE gel.
• Once electrophoresis is initiated, the DNA
fragments migrate out of the agar block and
directly into the gel