Restriction Fragments and Mapping Restriction Fragment Analysis System used to compare the genes and DNA sequences between individuals in a population. can be used to identify heterozygous carriers of mutant alleles Uses restriction enzymes to digest (break apart) DNA into shorter segments. DNA segments may differ in length based on the mutations present in genes restriction enzymes are specific for nucleotide sequences a change in the sequence may cause an enzyme not to make a cut resulting in a larger segment RFLPs (restriction length fragment polymorphisms) present non-coding sections of DNA used to identify different relatedness of individuals in a population can be used to construct linkage maps

RFLPs

Southern Blotting Segments can be compared to find differences by gel electrophoresis - southern blotting gel electrophoresis takes advantage of the overall negative charge associated with DNA molecules DNA digests are put into wells (holes in the gel) and guided through the gel by an applied electrical current gel acts as a molecular sieve (filter) allowing the smaller molecules to travel the furthest in a given amount of time fluorescent or radioactive markers are then added to the gel to elucidate the bands present

Linkage mapping by FISH (fluorescent in situ Hybridization) Fluorescent probes create a map of whole chromosomes as they hybridize with them. the distance between the individual fluorescent probes creates a map of the chromosome The physical map is then constructed as DNA digests are compared to find areas of overlap accomplished through cloning with YACs & BACs Once reconstructed the individual fragments can be fed through a sequencing machine to establish the nucleotide sequence

FISH

DNA Sequencing: The human Genome Project that spanned from 1993 to 2003 pushed the development of faster more efficient methods of DNA sequencing. Dideoxy Chain-Termination Method DNA to be sequenced is digested and amplified (phage vectors, YACs & BACs) Cloned fragments are then sequenced using the dideoxy method fragments are incubated in a test tube the following: primers DNA polymerase deoxyribonucleotides (normal DNA components) dideoxyribonucleotides with the addition of a dDNA elongation of the growing strand is terminated each different dDNA is fluorescently labeled with a different color ddATP - green ddCTP - blue ddTTP - red ddGTP - yellow the result is many strands of different lengths with different colored termination points the incubated fragments are then separated by weight and size through a polyacrylamide gel in a column (capillary tube) finally a sequencing machine gives the sequence based on the weight and the end marker of each strand