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?