Function: DNA is fragmented (digested; cut) at specific sites

Transcription

1 ENZYMES TOOLS FOR RECOMBINANT DNA TECHNOLOGY Restriction endonucleases (REs); Function: DNA is fragmented (digested; cut) at specific sites -found in bacteria and serve to degrade invading DNA -recognize specific base sequence that reads the same 5 to 3 on opposite strands; ex. Eco RI digests 5' GAATTC 3'/3'CTTAAG 5' between each G and A, leaving AATT tails (overhangs) i.e.: -some targets are only 4 bases long ("4-cutters"), some are 6, and a few are 8 bases long, but all recognize symmetrical palindromes. (The same sequence when read 5' to 3' on each strand) -some leave 4 base overhangs, some 2 base overhangs and some make blunt-ended cuts. -each bacterial species makes one or a few different restriction endonucleases that cut different target seqeunces - to prevent cutting their own DNA, the bacteria have another enzyme that adds a methyl group to one of the bases in the same sequence. Page 1 of 6

2 -for hundreds of examples, click on: DNA ligase; Function: Connect DNA fragments together; matching sticky ends make it easy to connect different fragments, but even blunt ends can be ligated at low efficiency Reverse transcriptase; Function: use mrna as a template to make a complimentary DNA strand (cdna) Phosphatase; ex CIAP; Function: remove a terminal PO4; unless at least one of the ends has a phosphate, two pieces of DNA cannot be ligated, even if they have complementary tails. After digesting a plasmid vector with a restriction nuclease that has a single cut site, treating with phosphatase will prevent the vector from re-annealing; only vectors where another piece of DNA has been inserted will form a stable, replicating circle. DNA polymerases; Function: make copies of target DNA All polymerases require a primer to get started; they can extend the primer by making base-pairs to match a template strand All polymerases add nucleotides in the 5' to 3' direction only Examples:. Taq polymerase is isolated from hot springs bacteria and can be used in thermal cyclers in a Polymerase Chain Reaction (PCR) for in-vitro amplification of DNA PolA, or Klenow fragment used to make radioactively labeled probes by incorporating 32 P-dATP etc. TdT (terminal deoxynucleotidyl transferase) adds tails of available base RNase H - makes nicks in an RNA strand hybridized to a DNA strand 2 DNA OLIGOMERS primers are short sequences of single stranded DNA often used to target DNA polymerase to a specific starting location Page 2 of 6

3 -a pair of primers-one complementary to bases on each strand of a DNA double helix- can be used to amplify DNA between them using TAQ polymerase and PCR oligo-dt can be attached to beads or resins for "pulling" eukaryotic mrna out of solution or to prime reverse transcriptase. linkers these have tails that overlap the tails produced by different restriction enzymes on each end, so can "link" different ends promoters can be added to vectors so that the cloned gene will be expressed in response to the appropriate environment (ex., lactose for the Lac operon promoter) or cell type signal sequences can be added to the beginning of messages to target a protein to selected organelles or for secretion. VECTORS: Tools for getting genes into and copied or expressed in different host cells. Plasmids or yeast) circles of DNA that can replicate in a host cell (usually E. coli puc plasmids that are often used to clone genes for replication and/or expression in E. coli have the following features: - approximately 3,000 base-pairs (bp) in a closed circle - an origin of replication (ori) that permits replication of over 70 copies per cell - a gene for ampicillin resistance to serve as a selectable marker, that is, host cells without the plasmid are killed by the antibiotic 3 ampicillin; only cells that contain the plasmid can survive in the presence of ampicillin - a " multiple cloning site" near the beginning of the lacz gene; the multiple cloning site (MCS) is a man-made sequence that includes up to 13 unique restriction endonuclease target sequences; it is also called a polylinker Page 3 of 6

4 the host cell does not have the lacz gene but does have the I gene, so that when grown on lactose, a message will be made from any DNA inserted into the MCS. this also permits "blue-white" screening since any bacterial colony with a rejoined plasmid (no insert) will make β- galactosidase and turn a compound added to the medium (Xgal) blue. If the Z gene is disrupted, the colonies are white. An example of a gene cloned into a puc is shown on the next page: 4 Page 4 of 6

5 5 Viruses portions of Lambda (an E. coli virus) or Adenovirus, (a human virus) can be removed and replaced with cloned genes without preventing the virus from infecting host cells. Cosmids vectors that have properties of a plasmid combined with the parts of a virus needed for putting the DNA in a virus coat BACS bacterial artificial chromosomes; very large circles of DNA that replicate more like a full sized chromosome than a plasmid YACs yeast artificial chromosomes; include centromeres, telomeres and origins so they will go through mitosis and meiosis in a yeast cell. Vector choice? Advantages: Disadvantages Page 5 of 6

6 PLASMID VIRUS COSMIDS BAC easy to isolate many copies/cell available for many cell types selectable markers can include expression larger inserts target host cells large inserts kb inserts Stable small insert (< 8,000 bp) must transform into bacterial cells; create pores via osmotic shock electric shock may need packaging may stimulate immune response require packaging repeated sequences included one copy per cell YAC huge inserts very tricky to handle rearrangement common may combine different fragments and rearrange 6 Page 6 of 6