restriction fragments and mapping

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