1. 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

2. Southern Blotting Segments can then be compared to find the 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

3. 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

4. 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