Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing...
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Transcript of Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing...
Cloning lab resultsCloning the human genomePhysical map of the chromosomesGenome sequencingIntegrating physical and recombination mapsPolymorphic DNA markers CF jumpingExample of a molecular forensics study
Research paper outlines: you can still email me your outline
Nov. 9, 2009
Cloning a gene into a plasmid for replication in E. coli
HindIII BamH1
CAT
Blunt ends are compatible with any other Blunt end
Sticky ends are only compatible with an end that leaves the complementary single-stranded overhang. Because sites are palindromic, sites cut with the same enzyme are compatible.
Single stranded overhang aids ligationpreligation complex lasts longer.
Restriction enzymes used in cloning experiments
pBKS BH
pCAT BH
pBKS Un
pCAT Un
Tuesday class gel results
Open circle
supercoil
Libraries of genome sequences or transcripts (cDNA)
Cut DNA into fragments,
Ligate to replication vectors
Random pieces of DNA are cloned into replication vectorswhere they can be accessed whenever needed.
Generate contigs that replicate DNA sequence of entire chromosomes
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 genesLow 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 sequencesBACS 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 markersWith disease. Find contigs that span markersCompare DNA sequence.
Forensics: Using DNA polymorphism to distinguish individuals
Need: Rapid and reliable markers
Sufficient numbers of polymorphic markersto 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 beinga parent.
With DNA technology,we have so many markers that the probability ofmistaken identity becomesvery small
Insertion-deletion polymorphism
Variable number tandem repeats-Minisatellites (10-100 bp repeats)
Short tandem repeats-Microsatellites (1-3 bp repeats)
Single nucleotide polymorphisms
Different types of DNA markers
Insertion-deletion polymorphism
Allele 1
Allele 2
Transposon, retrovirus, etc
Point of referenceRestriction sitePCR primer
Sobrino et al 2005 Forensics Sci. Int. 154: 181-194
VNTR
Variable Nucleotide Repeats
Up to 100bp repeats
VNTR or
Fig. 11.3
Microsatelites
1-3 bp repeats
Polymerase pauses,replication continuesout 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 identifiedin 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?