Manipulating DNA Genetic Engineering uses the understanding of the properties of DNA to study and...
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![Page 1: Manipulating DNA Genetic Engineering uses the understanding of the properties of DNA to study and change DNA sequences in living organisms – Invitro… in.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e1a5503460f94b07471/html5/thumbnails/1.jpg)
Manipulating DNA
• Genetic Engineering uses the understanding of the properties of DNA to study and change DNA sequences in living organisms– Invitro… in the lab– Invivo… in the organism
• The human Genome Project that spanned from 1993 to 2003 pushed the development of faster more efficient methods of DNA sequencing.
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Restriction Fragments and Mapping• Uses restriction
enzymes to digest (break apart) DNA into shorter segments. – 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
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Amplification of DNA using PCR• Polymerase Chain Reaction (PCR) is a
quicker method of amplifying small amounts than any other method available – genomic DNA is denatured with heat
and cut with restriction enzymes – primers specific to the desired DNA
attach to complimentary sequences – heat stable DNA polymerase
completes the complimentary strand – the process is then repeated making
billions of copies within a few hours
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Restriction Fragment Analysis• System used to compare the genes and DNA
sequences between individuals in a population.– Gel electrophoresis
• can be used to identify heterozygous carriers of mutant alleles – DNA segments may differ in length based on the mutations present in
genes
• Can be used to identify members of populations by inherited differences in DNA amount– 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
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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 view the bands present
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DNA Sequencing:• Dideoxy Chain-Termination Method
– Amplified 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
» each different dDNA is fluorescently labeled with a different color • ddATP - green • ddCTP - blue • ddTTP - red • ddGTP - yellow
– finally a sequencing machine gives the sequence based on the weight and the end marker of each strand