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In Vitro DNA Recombination Techniques and Requirements

Discover how DNA is recombined in test tubes using restriction enzymes and DNA ligase. Learn how to cut and ligate double-stranded DNA ends specifically, isolate and amplify recombinant DNA, and store it. Explore the discovery of restriction enzymes and their role in recombinant DNA technology. Visualize restriction fragments separated by gel electrophoresis and learn about DNA sequencing methods.

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In Vitro DNA Recombination Techniques and Requirements

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  1. How is DNA recombinedin test tubes (in vitro)? Requirements • How to cut double-stranded DNA specifically? • How to ligate double-stranded DNA ends specifically? • How to isolate specific “recombinant DNA”? And to amplify it? • How to store individual “recombinant DNA”?

  2. Discovery of restriction enzymes and their characterizations made “recombinant DNA” possible

  3. Enzymes that cut double-stranded DNA specifically – type II restriction endonucleases • specific restriction sites (recognition site with 4 ~ 6 bp is most commonly used) palindromic (or dyad symmetric) • generating 3 types of ends 1. sticky ends with 5’ overhang (or 3’ recessed) 2. sticky ends with 3’ overhang 3. blunt ends

  4. Restriction fragments with complementary “sticky ends” are ligated easily

  5. Visualization of restriction fragments separated by gel electrophoresis Ehtidium bromide staining UV from underneath

  6. Enzymes that ligate double-stranded DNA ends (without changes in bases) DNA ligase: ligating 3’-OH with 5’-P through an Enz-cofactor intermediate 1. E. coli DNA ligase: utilizing NAD+ as co-factor 2. T4 DNA ligase utilizing ATP as co-factor 3. eukaryotic DNA ligase utilizing ATP as co-factor

  7. NOT all ligated DNA are desiredeach is present in low amounts 1. cut and uncut vector DNA 2. unligated insert DNA 3. self-ligated vector DNA 4. ligated, but linear, vector + insert DNA 5. desired recombinant DNA, as circular DNA 6. and others Which could enter E. coli? Which is desired?

  8. E. coli serves as natural ‘factory’ for amplifying recombinant DNAand for their storage and isolation Introduction of recombinant DNA into E. coli – transformation requirements: 1. “competent” E. coli 2. “vector” for carrying desired insert DNA 3. ways for identifying and isolating cells containing desired recombinant DNA

  9. Requirements for E. coli to be competent • Pre-treatments (CaCl2 & “heat shock”) • Circular, but not linear, DNA • DNA with “origin of replication”

  10. Plasmids are extrachromosomal self-replicating DNA molecules • Suitable as vector: • small • has an ori • has genes as “selection marker” • has “unique” restriction sites pBR322 an artificial plasmid

  11. Suppose target DNA is inserted at PstI site

  12. How to isolate E. coliwith desired recombinant DNA • To distinguish “transformed” from “non-transformed” bacteria How? selection marker – antibiotic-resistance gene carried on vector DNA • To distinguish transformants with vector from those with recombinant DNA How? “screening” marker – antibiotic-resistance gene? or else?

  13. Polylinkers facilitate insertion of restriction fragments into plasmid vectors

  14. Source of insert DNA • chromosomal DNA by restriction enzyme digestion by PCR (polymerase chain reaction) others? • mRNA  cDNA by RT-PCR (reverse transcription + PCR) • chemically synthesized

  15. Synthesis of cDNA mRNA + reverse transcriptase + dNTPs  cDNA Does reverse transcriptase require primer for DNA synthesis?

  16. The polymerase chain reaction (PCR)

  17. Requirements for PCR amplification of DNA 1. template (very little) - double stranded DNA - single stranded cDNA (RT-PCR) 2. a pair of primers (oligonucleotides) one upstream, one downstream, each complementary to opposite strands 3. DNA polymerase that is resistant to high temperature (Taq DNA polymerase) 4. dNTPs

  18. Restriction enzyme site could be introduced into primers prior to PCR

  19. Small DNA molecules (oligonucleotides, or oligomers) can be chemically synthesized

  20. Improvements in DNA Technology • Development of Vectors: • Plasmid • l phage & other phages • Cosmid • Bacterial artificial chromosomes (BAC) • Yeast artificial chromosomes (YAC) • Procedures for introducing DNA into bacteria: • Transformation • in vitro packaging (l and cosmid only) • electroporation

  21. Selection of target clone from a “gene library” using hybridization

  22. DNA sequencing data

  23. DNA sequencing: the Sanger method(dideoxy chain termination method) 4 separate polymerization reactions are performed, each including a different ddNTPs

  24. DNA sequencing the Sanger (dideoxy) method Figure 7-29b,c

  25. DNA sequencing: the Maxam-Gilbert method(chemical degradation method)

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