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Recombinant DNA Technology (8)
• Cloning Eukaryotic DNAs in Phage Genomes
– Recombinant DNA molecules are packaged into
lambda phage heads.
• Lambda phage can take inserts up to a size of 25 kb.
• Sites of phage infection are identified as plaques in a
bacterial “lawn”.
• DNA fragments are identified by the same techniques
as those used for cloning of recombinant plasmids.
Protocol for cloning
eukaryotic DNA
fragments in lambda
phage
18.14 Enzymatic Amplification of DNA
by PCR (1)
• Polymerase chain reaction (PCR) is a
technique to amplify specific DNA fragments.
– It uses a very small amount of template.
– Utilizes a heat-stable DNA polymerase (Taq
polymerase) from bacteria living in hot springs.
– Uses repeated cycles of denaturation, DNA
replication, and cooling to double the amount of
DNA during each cycle.
– Uses an automated thermal cycler.
PCR
Enzymatic Amplification of DNA by
PCR (2)
• Applications of PCR
– Amplifying DNA for cloning or analysis—such as in
criminal investigations and fossil analysis.
– Testing for the presence of specific DNA sequences
– Comparing DNA molecules
– Quantifying DNA or RNA templates
18.15 DNA Sequencing (1)
• Techniques developed in the 1970s are widely
used for sequencing nucleic acids.
• The Sanger-Coulson dideoxy method became
the most widely used:
– Four samples of identical single-stranded DNA
molecules are obtained.
DNA Sequencing (2)
• Dideoxy sequencing (continued)
– DNA of each sample is incubated with a primer,
DNA polymerase, four dNTPs, and a low
concentration of ddNTPs (dideoxyribonucleoside
triphosphates), different one in each sample.
– DNA fragments of different lengths are
synthesized in each sample, with synthesis
terminating where ddNTP has been randomly
incorporated.
DNA Sequencing (3)
• Dideoxy sequencing (continued)
– DNA fragments are separated by gel
electrophoresis and the DNA sequence is read
form the gel bands.
– The amino acid sequence of the protein is
deduced from the nucleotide sequence.
DNA sequencing by the dideoxy method
Result form DNA sequencing
DNA Sequencing (4)
• Next-generation sequencing (NGS) is based on
polymerase-dependent DNA synthesis but
does not depend upon premature
termination.
– It identifies the individual nucleotides as they are
being incorporated by the polymerase in real time.
18.16 DNA Libraries (1)
• DNA libraries are often produced from DNA
cloning:
– Genomic libraries are produced from DNA
extracted from nuclei and contain all DNA
sequences of the species.
– cDNA libraries are derived from DNA copies of an
RNA population.
DNA Libraries (2)
• Genomic Libraries
– DNA fragments for the library can be obtained by
cutting genomic DNA with restriction enzymes.
– Cleaving of genomic DNA is random, which
generates overlapping fragments.
– Overlapping fragments are useful for chromosome
walking, to study linked sequences in an extended
region of a chromosome.
DNA Libraries (3)
• Cloning Larger DNA Fragments in Specialized
Cloning Vectors
– A yeast artificial chromosome (YAC) can
accommodate large (up to 1000 kb) DNA inserts.
– A bacterial artificial chromosome (BAC) accepts
DNA inserts of up to 500 kb, and can be quickly
grown to large numbers.
DNA Libraries (4)
• cDNA Libraries
– The isolation of genomic fragments allows study
of the genome.
– Coding regions of a gee can be studied using
cDNAs, synthesized by reverse transcriptase using
mRNA as a template.
– Allow foreign DNA to be transcribed and
translated during the infection process.
Synthesizing cDNAs for cloning in a plasmid
18.17 DNA Transfer into Eukaryotic
Cells and Mammalian Embryos (1)
• DNA incorporation into the genome of a
nonreplicating virus is called transduction.
• DNA introduced into cultured cells is called
transfection.
• The gene whose role is being investigated
after transfection is called a transgene.
DNA Transfer into Eukaryotic Cells and
Mammalian Embryos (2)
• A direct way to introduce foreign genes into a
cell is by microinjection of DNA directly into
the cell nucleus.
• Animals that have been genetically
engineered to that their chromosomes have
foreign genes are called transgenic animals.
Microinjection of DNA
Transgenic mice
DNA Transfer into Eukaryotic Cells
and Mammalian Embryos (3)
• Transgenic Plants and Animals
– Transgenic organisms allow scientists to
determine the effects of overexpression of a
particular DNA sequence.
– Genetic engineering can produce animal models
used to study human diseases.
– The main goal of genetic engineering in plants is
to improve the efficiency of both photosynthesis
and nitrogen fixation.
Formation of transgenic plants using the
Ti plasmid
18.18 Determining Eukaryotic Gene
Function by Gene Elimination (1)
• In Vitro Mutagenesis
– Site-directed mutagenesis (SDM) allows making
small changes in a DNA sequence.
– SDM is accomplished by synthesizing a DNA
containing the desired change and allowing it to
hybridize to a single-stranded normal DNA.
– The polymerase elongates the replicates DNA
adding nucleotides complementary to the normal
DNA.
Determining Eukaryotic Gene
Function by Gene Elimination (2)
• Knockout Mice
– Knockout mice are obtained by transfecting
embryonic stem cells, introducing them into an
embryo, and implanting the embryo into a female
mouse.
– Germ cells containing the knockout mutation are
heterozygous, which can be used to obtain a
homozygous mutant phenotype.
– Genes can be assessed for their functions.
Formation of knockout mice
Formation of knockout mice
Formation of knockout mice
Formation of knockout mice
Determining Eukaryotic Gene
Function by Gene Elimination (3)
• RNA Interference
– Specific mRNAs can be degraded in vivo by
treating with small double-stranded siRNA
containing part of the sequence of the target
mRNA.
– Cells treated with RNAi cannot make the protein
encoded in the target mRNA.
– Libraries containing thousands of siRNAs are
available for the study of gene function.
Determining gene function by RNA interference
18.19 The Use of Antibodies (1)
• Antibodies are highly specific proteins
produced by lymphoid tissues in response to
the presence of foreign materials.
• Preparation of antibodies:
– A population of polyclonal antibodies can be
obtained by repeated injections of a purified
antigen into an animal.
– The blood of the animal serves as a source of an
antiserum.
The Use of Antibodies (2)
• Preparation of antibodies (continued):
– A monoclonal antibody is produced by
descendants of a single antibody-producing cell:
• Antibody-producing cells do not grow and divide in
culture.
• Fusion of a normal antibody-producing lymphocyte and
a malignant myeloma cell will create a viable
hybridoma cell that can produce large amounts of a
monoclonal antibody.
Formation of
monoclonal
antibodies
The Use of Antibodies (3)
• Antibodies can be conjugated with a
fluorescent substance that allows visualization
of antigens.
– In direct immunofluorescence, antibodies with
bound fluorescent molecules bind to antigens and
can be visualized with a fluorecence microscope.
– In indirect immunofluorescence, cells are
incubated with unlabeled antibodies, and then
with labeled 2˚ antibody against the 1˚ antibody.