Fig 11-1 Chapter 11: recombinant DNA and related techniques

Recombinant (chimeric) DNA: fused DNA from two different organisms Recombinant clone: vector (bacterial plasmid, virus) + insert (DNA fragment to be cloned) Recombinant (transgenic) organisms: host genome + clone from another organism

cDNA: “complementary DNA”; DNA complementary to RNA Usually made against mRNA cDNA is essentially an intron-less copy of a gene, minus 5’ and 3’ flanking regulatory regions of the gene Prepared using reverse transcriptase (an RNA- dependent DNA polymerase enzyme of RNA viruses)

Fig 11-2 Creating cDNA (DNA complementary to mRNA)

Fig 11-2 Creating cDNA (DNA complementary to mRNA)

Fig 11-2 Creating cDNA (DNA complementary to mRNA)

Fig 11-2 Creating cDNA (DNA complementary to mRNA) Creates clonable DNA copy of specific mRNA or can make cDNA library (representing mRNA population)

Fig 11-3 Using restriction sites to create a recombinant molecule

Fig 11-4

pallindromic sequence cohesive ends

Fig 11-5 Using restriction sites to create a recombinant molecule

Fig 11-5 Using restriction sites to create a recombinant molecule

Fig 11-6 Cells receiving a complete plasmid form colony Grow and purify DNA from single colony Useful for inserts <10kb

Fig 11-6 Using antibiotic resistance markers to select plasmid-bearing colonies

Bacteriophage lambda: engineered as vector for cloning large DNA fragments Central 1/3 of genome (~45 kb) contains lysogenic function genes Can substitute ~15 kb cloned DNA into genome and the virus is still capable of lytic infection e.g., the Drosophila genome (~150,000 kb) can be contained in a minimum of 10,000 recombinant lambda clones (can fit on one 15 cm Petri plate)

Fig 11-7 Creating a genomic library in bacteriophage lambda

Fig 11-7 Creating a genomic library in bacteriophage lambda Useful for inserts 10-20kb

Fig 11-8 Useful for inserts kb

Fig 11-9

Identifying a desired clone/gene in a library: Use a probe (previously cloned DNA, oligonucleotide, or antibody)

Fig Detecting & isolating a specific clone within a library by hybridization

Fig 11-1 Using an antibody to detect & isolate a specific clone within a library

Identifying a desired clone/gene in a library: Use a probe (previously cloned DNA, oligonucleotide, or antibody) Functional complementation (useful in organisms with small genomes) Positional cloning (chromosome “walk” to mutant rearrangement site)

Fig Chromosome walking to identify/isolate a region containing a gene

Fig Agarose gel electrophoresis separates DNA fragments by size: restrict cloned DNA electrophoresis stain with ethidium bromide visualize under UV

Fig Southern/Northern blot analysis agarose gel electrophoresis transfer to nitrocellulose hybridize with radioactive probe autoradiograph to detect bands containing probe sequence

Fig Using restriction sites as markers to map a DNA fragment

Fig Using restriction sites as markers to map a DNA fragment

Fig Dideoxynucleotide used for Sanger DNA sequencing

Fig Sanger dideoxy DNA sequencing

Fig Sanger dideoxy DNA sequencing Mixture of ddATP + dATP permits formation of chains of various lengths common 5’ end (primer) vary by 3’ ends, marking locations of A residues (T residues on template)

Fig Sanger dideoxy DNA sequencing

Fig Sanger dideoxy DNA sequencing

Fig Automated sequencing readout of Sanger dideoxy DNA sequencing

Fig An initial bioinformatic analysis Scan sequence for exceptionally long ORFs

Polymerase chain reaction (PCR) Uses heat-stable DNA polymerase (e.g., Taq polymerase) Requires two opposite-strand primers; ~100 bp - ~3 kb apart on the target template Uses a regimen of temperature cycling to amplify the DNA target between the two primers

Fig Polymerase chain reaction Specific primers permit specific amplification of a DNA segment

Fig Understanding alkaptonuria

Fig Detecting sickle-cell β–globin allele

Fig Detecting sickle-cell β–globin allele Heterozygote?

Fig Ti plasmid: a vehicle for making transgenic plants

Fig 11-29

Fig 11-30

Fig Inherited as a Mendelian dominant marker

Engineering of mammalian genomes Insert a gene (relatively easy) Destroy a gene (“knockout”) Replace a gene (e.g., gene therapy)

Insertions at random (ectopic) sites Ectopic transformation of mouse embryos Fig 11-34

Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

Making a targeted mutation (“knockout”) in mouse cells Fig 11-35

Fig Using embryonic stem cells to make a knockout mouse

Fig Using embryonic stem cells to make a knockout mouse

Gene replacement therapy of lit mice Fig 11-38

Gene replacement therapy of lit mice Fig 11-38

Complications arising with germline gene therapy to cure genetic diseases in mammals is that most transgene integration events are random (not targeted) Transgene does not replace defective gene (just complements it) Transgene insert might disrupt another gene (creating an undesired mutation) Transgene will usually segregate independently from the disease-causing gene

Alternatives in gene therapy Fig e.g., transgene on viral vector

Fig 11-