Bio3400 Chapter 19 Recombinant DNA Technology
  1. A              enzyme binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction fragments.

      Restriction enzymes such as BamH1 (from Bacillus amyloliquefaciens H) bind to and cut DNA at specific sites (recognition sequences) as a defense mechanism against infection by viruses.

      Bacterial restriction enzymes cut DNA at recognition sequence, producing complementary sticky ends. Enzymes with a four-base recognition sequence such as TaqI will cut, on average, every 256 (4^4) base pairs. EcoRI has a six-base recognition sequence,, and produce average fragments of 4096 (4^6) base pairs.
  2. Most recognition sequences are              , and restriction enzymes can cleave these sequences in an offset manner to produce         ends.

      The restriction enzyme EcoRI recognizes the palindromic nucleotide sequence GAATTC. Cleavage of DNA at this sequence produces complementary single-stranded tails. These complementary tails are "sticky ends" that can anneal with each other, even if the two fragments are from different sources.

      Restriction fragments from different sources is cleaved with EcoRI and mixed to allow annealing of the sticky ends. DNA ligase then chemically bonds these annealed fragments into recombinant DNA.
  3. DNA fragments can be         using restriction enzymes and          such as plasmids and then replicated in host cells.

      DNA cloning can be done by cutting a plasmid and the target DNA with the same restriction enzyme. The target DNA is spliced into the vector and transferred to a host for replication. Recombinant cells are identified by growth on selective medium and isolated. Vid/Klug8e/ch19/19_2_2a.swf

      A plasmid is a double-stranded DNA molecule in bacterial cells that replicates independent of the host chromosome. Plasmids such as these isolated from E. coli can be used as vectors for carrying restriction fragments into host cells for cloning DNA.

      The plasmid pUC18 contains an antibiotic resistance gene to screen for host cells that have taken up vectors. A polylinker region contains many restriction enzyme recognition sequences. The polylinker is inserted into a lacZ gene, whose gene product b-galactosidase can metabolize Xgal supplied in the medium; bacteria with wild type lacZ produce blue colonies. Recombination within the polylinker disrupts lacZ function, resulting in white colonies.

      Bacterial cells that have taken up plasmids containing the ampicilin resistance gene can form colonies in a Petri dish containing ampicilin and Xgal. Cells with recombinant polylinker have a defective lacZ gene, making them unable to digest Xgal and form white colonies instead of blue. Plasmids can carry up to 10 kb of recombinant DNA.
  4. Other          that can carry larger target DNA include phages, cosmids, and            artificial chromosomes (BACs).

      Phage l (lambda) can be used as a cloning vector. The foreign DNA to be cloned (up to 20 kb) is ligated into the phage DNA with a restriction enzyme. The recombinant chromosome is then packaged into phage proteins to form a recombinant virus.

      The bacteriophage l (lambda) possesses a linear double-stranded DNA molecule in its protein coat, which closes to form a ring upon infection of the host cell, as seen in the electron micrograph at right.

      Cosmids are created by combining parts of the lambda chromosome with parts of plasmids. The cosmid pJB8 contains:
    • an origin of replication (ori) that allows it to replicate as a bacterial plasmid,
    • a cos gene for packing phage DNA into protein coats,
    • an ampicillin resistance gene (amp),
    • a region containing restriction sites for cloning (BamHI, EcoRI, ClaI, and HindIII). Cosmids can carry up to 50 kb of inserted DNA.

      A bacterial artificial chromosome (BAC) ia a vector with a large cloning capacity (up to 300 kb). A BAC is constructed from a fertility plasmid (F factor) that enables conjugation. A chloramphenicol resistance gene serves to screen for recombinants, while a polylinker contains several restriction enzyme recognition sites for inserting foreign DNA. Promoter regions at T7 and Sp6 allow expression of inserted genes.

      1. In the F^+, the two strands of the double helix of the Fertility (F) factor (a plasmid) separate. 2. One of the two strands moves into the recipient (F^-) cell. 3. The other strand remains in the donor cell. 4. Both strands are replicated, with clockwise rotation of the circles. 5. Both the donor and the recipient cells are now F^+ after conjugation.
  5. Often the cloned gene of interest should be             into proteins.             vectors are engineered to express large quantities of the encoded protein.

      pET is an expression vector engineered to produce copies of a selected protein in a host cell. A viral T7 RNA polymerase gene is controlled by a lac repressor (lacI) and operator (lacO), making it inducible by the lactose analog IPTG. Growing host cells on IPTG derepresses the T7 RNA polymerase gene. The RNA polymerase binds to its promoter and transcribes the target gene.

      When lactose is present, it indirectly induces the activation of the genes by binding with the repressor, which becomes inactive, and the operon is turned on.
  6. Eukaryotic hosts such as        cells are useful to produce eukaryotic proteins since they can perform                    modifications and carry very large DNA fragments.

      This yeast artificial chromosome pYAC3 contains telomere sequences (TEL), a centromere (CEN4, from chromosome 4), necessary for eukaryotic chromosomes. and many gene markers. Treatment with the restriction enzymes SnaB1 and BamH1 breaks it into two arms, and target DNA fragments ligated between the arms are cloned in yeast host cells. YACs can carry up to 1,000 kb of DNA, and can also carry out posttranslational modifications that eukaryotic proteins require.
  7. Bacterial plasmids can be used to transfer genes to plant cells in a process caled                 .

      A tumor-inducing (Ti) plasmid of the bacterium Agrobacterium tumifaciens can be used as a vector for DNA cloning in plants. The plasmid T DNA, necessary for integration, are combined with bacterial DNA that contain an origin of replication (ori), restriction sites and antibiotic resistance genes (Kan^R and tet^R). When the bacterium infects a plant, the T DNA, together with any cloned DNA, integrates into a host chromosome, producing a transgenic plant. This process is caled transformation.
  8. YACs can be used to transfer genes to animal cells in a process caled direct            .

      Cloned DNA carried by vectors such as YACs can be transferred in mammals by direct injection of purified recombinant DNA from a gel into the nucleus of oocytes. Transgenic zygotes are then implanted in foster mothers to develop into transgenic mice.
  9. The             chain reaction (PCR) can be used to amplify small quantities of target DNA in vitro.

      Kary Mullis (Nobel 1993) developed the Polymerase Chain Reaction (PCR) to amplify the quantity of target DNA in vitro.
    • The target DNA is denatured into single strands.
    • Each strand is annealed to a short, complementary primer.
    • DNA polymerase and nucleotides extend the primers, using the single-stranded DNA as a template. After one cycle of amplification, the amount of target DNA has been doubled.

      This cycle can be repeated several times. Each cycle of amplification doubles the amount of target DNA. 20 cycles can amplify the target DNA by a millionfold (2^20).
  10. Genes from whole chromosomes can be cloned to form genomic            . The individual chromosomes can be isolated by       cytometry and pulsed-field gel electrophoresis.

      Flow cytometry can sort metaphase chromosomes by staining with two fluorescent dyes, one that binds to AT pairs, the other to GC pairs. The stained chromosomes flow past a laser beam, and a photometer sorts them by differences in light scattering. DNA from the chromosomes can then be cloned and stored in genomic libraries.

      Intact yeast chromosomes can be separated using pulsed field gel electrophoresis, where the the voltage is periodically switched among 3 directions. This technique can separate very large DNA pieces such as 15 of the 16 yeast chromosomes shown here, with the largest chromosomes at the top. DNA from the chromosomes can then be cloned and stored in genomic libraries.

      Gel Electrophoresis. DNA fragments migrating through an agarose gel medium are separated by size, with the smallest pieces moving farthest. The fragments are stained with ethidium bromide and appear as bands when viewed under ultraviolet light.
  11. Libraries of transcriptionally active genes in particular cells can be constructed from                DNA (cDNA).

      Producing cDNA from mRNA. A short oligo-dT annealed to the poly-A 3' tail of mRNA serves as a primer. Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand (cDNA) and forms an RNA/DNA double-stranded duplex. Continue

      The RNA is digested with RNAse H, producing RNA fragments that serve as primers for DNA polymerase I to synthesize a second DNA strand. After sealing the gaps with DNA ligase, the result is a double-stranded cDNA molecule, free of introns.
  12. Specific clones can be recovered from a library by              with a probe followed by                  .

      Screening a plasmid library to recover a cloned gene.
    • Colonies of a plasmid library is overlaid with a DNA binding filter.
    • Colonies are transferred to the filter, then lysed, and DNA is denatured.
    • Filter is placed in a bag with a solution containing a radioactively labeled single-stranded DNA probe; the probe hybridizes with complementary DNA on the filter. Continue

    • Hybrids on the filter are detected by exposing it to X-ray film (autoradiography).
    • Colonies containing the hybrids are identified from the orientation of the spots.
    • Cells are picked from the colony for growth and further analysis.
  13. Specific DNA sequences can be identified with a           blot and autoradiography.

      Southern blotting can be used to identify DNA clones.
    • DNA samples are cut with restriction enzymes and separated by gel electrophoresis.
    • DNA is denatured and the gel placed on a sponge wick over a buffer.
    • DNA passes up from buffer to DNA-binding filter by capillary action. Continue

      Southern blotting.
    • DNA on the filter is hybridized with radioactive probes that bind to complementary sequences.
    • X-ray film is placed over the filter for autoradiography to identify the fragments.
  14. DNA fragments can be sequenced by          chain termination. Large-scale genome sequencing can be automated by using              dideoxynucleotides.

      DNA (Sanger) sequencing using dideoxy chain termination.
    • A primer is annealed to the target template DNA.
    • A mixture with DNA polymerase, the four dNTPs, and one dideoxynucleotide (such as ddATP), is added.
    • During primer extension, the polymerase occasionally inserts a ddNTP instead of a dNTP, which lacks the 3'-OH needed to attach the next nucleotide, terminating the chain.
    • Gel electrophoresis separates the synthesized fragments that terminate at the dideoxynucleotide.

      DNA sequencing by dideoxy chain termination. A gel showing fragments synthesized from four different dideoxynucleotide chain termination runs. The bottom band in the T lane are 5'-T and 5'-TT fragments, while the bottom band in the A lane is 5'-TTCGTGA.

      DNA sequencing using fluorescent dideoxynucleotides for chain termination. All four ddNTPs, labeled with fluorescent dyes, are used at the same time during primer extension.

      The products of the dideoxy chain termination reaction are analyzed on a single lane on a gel, and the bands are read by a fluorescence detector. This synthesized sequence begins with 5'-CTAGACATG.