restriction fragments and mapping
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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