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 ofspecific mRNA or

can make cDNA library (representing mRNA population)

Fig 11-3

Using restriction sitesto create a

recombinant molecule

Fig 11-4

Fig 11-4

4-6

4-4

pallindromic sequence cohesive ends

Fig 11-5

Using restriction sitesto create a

recombinant molecule

Fig 11-5

Using restriction sitesto create a

recombinant molecule

Fig 11-6

Cells receiving a completeplasmid form colony

Grow and purify DNAfrom 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 11-8Useful for inserts 100-300kb

Fig 11-9

Identifying a desired clone/gene in a library:

• Use a probe (previously cloned DNA, oligonucleotide, or antibody)

Fig 11-11

Detecting & isolatinga specific clonewithin a libraryby hybridization

Fig 11-1

Using an antibody todetect & isolatea specific clonewithin 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 11-15

Chromosome walking to identify/isolate a region containing a gene

Fig 11-13

Agarose gel electrophoresis separates DNA fragments by size:

• restrict cloned DNA

• electrophoresis

• stain with ethidium bromide

• visualize under UV

Fig 11-14

Southern/Northern blot analysis

• agarose gel electrophoresis

• transfer to nitrocellulose

• hybridize with radioactive probe

• autoradiograph to detect bands containing probe sequence

Fig 11-16

Using restriction sites as markers to map a DNA fragment

Fig 11-16

Using restriction sites as markers to map a DNA fragment

Fig 11-17

Dideoxynucleotide used for Sanger DNA sequencing

Fig 11-18

Sanger dideoxy DNA sequencing

Fig 11-18

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 11-18

Sanger dideoxy DNA sequencing

Fig 11-18

Sanger dideoxy DNA sequencing

Fig 11-19

Automated sequencing readout ofSanger dideoxy DNA sequencing

Fig 11-20

An initial bioinformatic analysisScan 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 11-21

Polymerase chain reaction

Specific primers permitspecific amplification of

a DNA segment

Fig 11-22

Understanding alkaptonuria

Fig 11-24

Detecting sickle-cell β–globin allele

Fig 11-24

Detecting sickle-cell β–globin allele

Heterozygote?

Fig 11-28

Ti plasmid: a vehicle for making transgenic plants

Fig 11-29

Fig 11-30

Fig 11-31Inherited 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 11-36

Using embryonic stem cells to make a knockout mouse

Fig 11-36

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 11-39e.g., transgene on viral vector

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