1 / 19

Study Skills: Recombinant DNA

Study Skills: Recombinant DNA . The Amoebas . Organized Concepts. Cutting and Pasting -Restriction Enzymes and how they work -Plasmids (vectors) -How Restriction enzymes and plasmids interact Transformation -General process and the origin of replication Analyze -gel electrophoresis

onan
Download Presentation

Study Skills: Recombinant DNA

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Study Skills: Recombinant DNA The Amoebas

  2. Organized Concepts • Cutting and Pasting • -Restriction Enzymes and how they work • -Plasmids (vectors) • -How Restriction enzymes and plasmids interact • Transformation • -General process and the origin of replication • Analyze -gel electrophoresis -heterozygous vs. homozygous -

  3. Cutting: Restriction Enzymes • Restriction enzymes cut DNA at a specific recognition nucleotide sequence known as a restriction site. They must be read in the 5’-3’ mannor. • make two incisions, once through each sugar-phosphate backbone of the DNA double helix. • They come from bacteria, where they are used to fight against viruses. • The specific cuts they make are useful for the first step in forming recombinant DNA.

  4. Pasting: Plasmids (Vectors) • A plasmid is a spherical DNA sequence found in bacteria The plasmid consists of… • An origin of replication, which allows it to replicate. • Aresistance gene against an antibiotic (ampicillin for example) • Promoters • About 4,000-10,000 base pairs long.

  5. Cutting and Pasting • Example: EcoRI(restriction enzyme from E.Coli) • This cut can be made in a spherical vector, plasmid, which will make it linear. • Two EcoR1 cuts are made in a donor DNA sequence, producing a fragment that will fit between the open ends of the plasmid. • The plasmid sequence and new DNA sequence will hydrolyze, and DNA ligase will seal the bonds. Making a recombinant plasmid.

  6. Cutting and pasting continued • Many different restriction enzymes can make cuts, thus variations of new DNA sequences can be ligated into the plasmid.

  7. Cutting + Pasting: Thoughtful Question • In real world, can two restriction enzymes cut the DNA at the same spot in an organism? • If the first one is possible, Will two restriction enzymes cutting the DNA in the same spot but two different ways, be considered two fragments DNA?

  8. Transformation: Origin of replication • Recombinant plasmids are placed into bacteria, E. Coli, for example. • The bacteria containing antibiotic resistant, recombinant plasmids, are then placed into the antibiotic they are resistant to. • Those containing DNA will begin to replicate and form large colonies • The replication occurs due to the origin of replication • plasmid detects the antibiotic and the promoter is signaled. • replication of the recombinant plasmid begins. Thus purifying the DNA fragment. • The origin of replication is species specific • Ex: A human origin of replication won’t function in E.coli, which is a bacteria.

  9. Transformation: Thoughtful Question • Is it possible to incorporate genes from other species, even as specie specific as they may be, into the human genome where we would be able to produce the protein and gain a desired trait?

  10. Analyze: Gel electrophoresis • locates (or amplifies) a certain DNA fragment on the gel which can be compared to another. • Shows which restriction enzymes produce the largest fragments • There are lanes made for each fragment produced by specific restriction enzymes. • The First band made at the top of the gel, signifies the length of the plasmid. • Bands appearing below that, show where there is a cut, and thus the length of a fragment. • Fragments are measured in base pairs. • Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. Ex. Question: Which two restriction enzymes in the following ex. gel produce the longest fragment?

  11. Analyze: Heterozygous vs. Homozygous • When determining strands based off of restriction sites, pay attention to the genotype of the specimen because a heterozygote would display all the possible strand lengths • Example problem: • The ecoRI-HindIIIfragmentsare recessive, while the BamHI-HindIIIfragments are dominant, what are the possible fragments that can be made in a heterozygous organism?

  12. Analyze: Thoughtful Questions • When deciphering gel electrophoresis is it possible that even if an incorrectly inserted sample can display the predicted results? If not is there away to fix or determine if it was incorrectly placed before conducting the experiment? • Granted that a heterozygote would display all the possible strand lengths due to having both alleles, what would occur when a mutation was present in the allele? How would the determination of the strand lengths differ if it would?

  13. Conclusion • Key Notes

More Related