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Nov. 9, 2009
Cloning lab results
Cloning the human genome
Physical map of the chromosomes
Genome sequencing
Integrating physical and recombination maps
Polymorphic DNA markers
CF jumping
Example of a molecular forensics study
Research paper outlines: you can still email me your outline
Cloning a gene into a plasmid
for replication in E. coli
HindIII
CAT
BamH1
Restriction enzymes used in
cloning experiments
Blunt ends are compatible with any other
Blunt end
Sticky ends are only compatible with an
end that leaves the complementary singlestranded overhang.
Because sites are palindromic, sites cut with
the same enzyme are compatible.
Single stranded overhang aids ligation
preligation complex lasts longer.
Tuesday class gel results
Open circle
supercoil
Libraries of genome sequences
or transcripts (cDNA)
Random pieces of DNA are cloned into replication vectors
where they can be accessed whenever needed.
Cut DNA into fragments,
Ligate to replication vectors
Generate contigs that replicate DNA sequence of entire chromosom
To make overlapping contigs,
DNA is partially digested with restriction enzymes
or physically sheared
Different kinds of replication vectors are available
for maintaining cloned DNA fragments in
bacteria or yeast
Choice of vectors depends in part on the desired size of the cloned DNA inserts
High copy number plasmids for inserts the size of single genes
Low copy number plasmids for larger inserts - genome fragments
Use end probes and fingerprinting to generate contigs
Fig. 10.8
Fig. 10.11
Combination of mapped polymorphic sequences and
genomic DNA clones enables reconstruction of
chromosome sequence
STS are polymorphic DNA sequences
BACS are cloning vectors with genomic DNA inserts
Polymorphic DNA markers allow association of
Phenotype with position on DNA map
Linkage with a family, high lod score of DNA markers
With disease. Find contigs that span markers
Compare DNA sequence.
Forensics: Using DNA polymorphism to distinguish individuals
Need: Rapid and reliable markers
Sufficient numbers of polymorphic markers
to be sure that no other person could be a match
DNA amplification methods to obtain information
from samples that are:
very small
have DNA degradation
Example of
paternity test
With this information,
we can only exclude
the accused from being
a parent.
With DNA technology,
we have so many markers
that the probability of
mistaken identity becomes
very small
Different types of DNA markers
Insertion-deletion polymorphism
Variable number tandem repeatsMinisatellites (10-100 bp repeats)
Short tandem repeatsMicrosatellites (1-3 bp repeats)
Single nucleotide polymorphisms
Insertion-deletion polymorphism
Allele 1
Transposon, retrovirus, etc
Allele 2
Point of reference
Restriction site
PCR primer
Sobrino et al 2005 Forensics Sci. Int. 154: 181-194
VNTR
Variable Nucleotide Repeats
VNTR or
Up to 100bp repeats
Fig. 11.3
Microsatelites
1-3 bp repeats
Polymerase pauses,
replication continues
out of register
microarrays
Some single nucleotide polymorphisms (SNP) can be detected as
RFLPs if they affect a restriction enzyme sequences.
Any single nucleotide polymorphism can be detected by
modern technologies such as oligonucleotide arrays
PCR can ease identification of
polymorphic sequences
and can be used to analyse very small
amounts of DNA
PCR copies
template
exponentially
Microsatellites or minisatellites can be detected by PCR
Use PCR to amplify sequences starting with
primers designed from single copy sequence that
flanks repeats
Genetic variation in a sequence with a
minisatellite used in DNA typing
Combining the information about the alleles identified
in a person for many loci (or many polymorphic markers)
can generate a unique pattern for every person.
Thomas Jefferson paternity investigation
Who’s Y chromosome is in the
male offspring of Sally
Hemings children?
Sally Hemings was
Jefferson’s slave and cared for
him late in his life. She had 5
children. After he died, she
claimed Jefferson was the
father of her children.
Jefferson was almost
impeached, charged with
being the father of Sally’s first
son, Thomas Woodson.
Jefferson denied that he was
the father. Forster et al 1988
used Y chromosome DNA to
see who was telling the truth.
Y chromosome
sequences are
highly conserved.
Comparing the
pattern of many
polymorphic
markers on the Y
chromosomes
makes it possible to
identify the lineage
of Y chromosomes
in modern
descendents of Sally
Hemings
Thomas Woodson
was Sally Hemings
first son. Eston
Jefferson was her
youngest son.
John Carr was the
brother in-law of
Thomas Jefferson.
Which lineage
matches the Y
chromosomes of
Eston Jefferson?
Thomas Woodson?