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. RFLPs

3. 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

4. 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

5. FISH

6. 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