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.

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

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

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

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

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