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 BamH1 CAT
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 single- stranded 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 pBKS Un pCAT BH pCAT Un pBKS BH 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
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
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 repeats- Minisatellites (10-100 bp repeats) Short tandem repeats- Microsatellites (1-3 bp repeats) Single nucleotide polymorphisms
Insertion-deletion polymorphism Transposon, retrovirus, etc Allele 1 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
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?