Cloning paper plasmid

CLONING PAPER PLASMID

Aim

  • To apply your knowledge of the “genetic code” in the making of a RECOMBINANT vaccine against smallpox and hepatitis B
    • To visually demonstrate recombination of DNA in the making of vaccines
    • To understand how genes can be inserted into another DNA – “recombine”
    • To conceptualize “restriction enzymes” and recognition of specific sites

Materials

  • DNAs to be combined carry other DNAs. This DNA will allow the recombinant DNA molecule (that you will make) to be duplicated in the bacterial host, Escherichia coli
  • VACCINIA VIRUS containing genes which code for surface proteins. These proteins will act as antigens against which antibodies can be formed by the person immunized. (Smallpox virus is so closely related to Vaccinia that Vaccinia virus is what is given in the traditional smallpox vaccination.) This DNA must be kept intact so it can serve as vector in a mammalian host.
  • HEPATITIS B VIRUS containing the gene for the major surface antigen (HBsAg). By inserting this gene into the vaccinia virus DNA, immunity may be elicited for both smallpox and hepatitis.
  • DNA LIGASE the enzyme which joins or “ligates” pieces of DNA together (use Scotch tape).
  • RESTRICTION ENDONUCLEASES to cut DNA at specific sites to make the pieces we need. Use scissors to “NICK” double stranded DNA on both strands. After being nicked, the strands are held together only by weak hydrogen bonds between complementary A-T and G-C pairs, fall apart and the DNA is broken. These enzymes are found in nature and used by bacteria to cut up and destroy “foreign” DNA. The unique ability to cut DNA only at specific nucleotide sequences have made these enzymes the cornerstone of the field of genetic engineering.
  • RESTRICTION ENDONUCLEASES: DNA SITES AT WHICH “NICKS” ARE MADE
    • BamHI G0GATC C FOR …G GATCC…
    • C CTAG=/G …CCTAG G…
    • SstI G AGCT0C …GAGCT C…
    • C=/TCGA G .. . C TCGAG…
    • HindIII A0AGCT T …A AGCTT…
    • T TCGA=/A …TTCGA A..
    • EcoRI G0AATT C …G AATTC…
    • C TTAA=/G …CTTAA G…
  • In this exercise, you are given three strips of paper representing a plasmid DNA (pBR322), a gene from Vaccinia and gene from Hepatitis.

Procedure

  • Isolate (cut out) the pBR322 DNA and circularize it into a small plasmid by using tape to connect the free ends. Be sure to make “staggered” cuts to preserve the “sticky ends”. Plasmids are circular, double –stranded extra chromosomal DNA molecules that contain specialized genes and have the ability to be replicated in the bacterial cell.
  • Isolate (cut out) the Vaccinia DNA fragment. Examine the DNA sequence for restriction enzymes that can be used to cut the vector pBR322.
  • Identify the restriction endonuclease used to generated the Vaccinia virus DNA fragment. Cut the vector, pBR322 with the same enzyme. BE SURE TO PAY ATTENTION TO WHERE THE ENZYME ACTUALLY NICKS THE STRANDS OF DNA TO GENERATE STICKY ENDS. Make sure the “sticky ends” of the DNAs are complementary A-T, C-G.
  • Ligate (scotch tape) the Vaccinia DNA and the Plasmid pBr322 vector. You NOW have a recombinant DNA plasmid that codes for the surface proteins of the virus.
  • Isolate (cut out) the Hepatitis B DNA fragment. You will now insert the hepatitis B DNA into Vaccinia virus DNA segment of your first recombinant DNA molecule.
  • Identify a restriction enzyme that can be used to cut out the HBsAg gene and insert it into your recombinant DNA MOLECULE RECOGNIZED BY A SPECIFIC RESTRICTION ENZYME, IT WILL CUT BOTH.
  • Ligate (Scotch tape) the hepatitis B DNA fragment to the Vaccinia virus DNA. You NOW have large circular molecule with DNA from three different sources. COMPARE your paper model with other students’. Discuss any differences.
  • How can this recombinant DNA molecule be used to generate a recombinant vaccine? What is the next step necessary in order for this new gene combination to be expressed?

PINK: pBR322 DNA with “Sticky Ends”

  • AmpR
  • Hind III
  • EcoRI
  • CCGAAGCTTCACGTAGCGCTAGGGCTAGGTAGCTTGCCATGGATTGAATTCGTGT
  • ACT
  • GCTTTACGGCTAAA
  • TTTGGCT
  • TCGAAGTGATCGCGATCCCGATCCATCGAACGGTACCTAACTTAAGCACATGAT
  • CGAA
  • ATGCCGA
  • “sticky ends”
  • PBR 322 DNA with “STICKY ENDS”
  • Use the “sticky ends” and scotch tape “LIGASE’ to make this PLASMID into piece of CLOSED
  • CIRCULAR DNA. The region encoding AMPR is marked by double lines.

YELLOW: Vaccinia DNA Fragment

  • HindIII SstI Bam HI Hind III
  • AGCTTATCGATCGGATTCGATCTAACGAGCTCATTTAGGCAGTCGAGTCCAATCG
  • ATG
  • GGATCCTACCGTA
  • ATAGCTAGCCTAAGCTAGATTGCTCGAGTAAATCCGTCAGCTCAGGTTAGCTACG
  • CCT
  • GGATGGCATTCGA
  • This represents a fragment of Vaccinia virus DNA which has been previously cut on each end with one of the restriction endonucleases with which you have been “supplied”. The Vaccinia surface protein region is marked by double lines.

GREEN: Hepatitis B Virus DNA Fragment

  • Bam HI SstI HBsAg Bam HI
  • GATCCAGTTGAGCTCTATGGCGATGACTACCAGTCTCAGAG
  • GTCAACTCGAGATACCGCTACTGATGGTCAGAGTCTCCTAG
  • This represents a fragment of the hepatitis B virus DNA containing the gene for the major surface antigen (HBsAg). This DNA has been previously cut with one of the restriction endonucleases with which you have been “supplied.” The HBsAg gene region is marked by double lines.
Last modified: Wednesday, 20 June 2012, 6:30 AM