Download Notes Biotechnology Chpt 20

DNA TECHNOLOGY
Chapter 20
BIOTECHNOLOGY
• The manipulation of organisms or
their components to perform tasks
• Genetic Engineering –
manipulation of genes
• Gene cloning – making identical
copies of a specific gene (a
specific nucleotide sequence)
Techniques we will use in our labs
• Transformation – putting a gene into bacteria
(Luc gene into E. coli to make them glow)
• Isolation of DNA – from cheek cells
• PCR – used to amplify a section of DNA (taster
gene now and later mtDNA)
• Restriction Enzyme Digest – use of enzymes
to cut DNA (plasmid mapping and taster gene)
• Gel electrophoresis – used to separate
different sizes of DNA fragments (plasmid
mapping, taster gene, and later mtDNA)
• Sequencing – determine exact base sequence of
a section of DNA (later with mtDNA)
Figure 20.1 An overview of how bacterial plasmids are used to clone genes
OVERVIEW OF GENE CLONING
• Isolation of plasmid DNA and gene to be
cloned
• Restriction enzyme digest
• Gene inserted into plasmid
• Plasmid put into bacteria
• Cells cloned with gene as part of plasmid
• Identification of desired clone
• Various applications
– Copies of protein product isolated
– Copies of gene transferred to another cell
Cutting DNA
• In nature, bacteria cut intruding (foreign) DNA
with restriction enzymes
• Enzymes cut at specific nucleotide sequences.
• Recognize their own DNA by methyl (-CH3)
groups added to adenines and cytosines
• Restriction Site – specific sequence that is
recognized by a specific restriction enzyme
• Cuts the phosphodiester bonds (breaks
backbone)
• Most sites are symmetrical:
GAATTC
CTTAAG
Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA
• Most restriction enzymes cut in a staggered
manner
G
AATTC
CTTAA
G
• The ends are called “sticky” because they are
complementary and would stick together
• Additional DNA with same sticky ends (cut
with same restriction enzyme) can be inserted.
• Ligase added to make the needed
phosphodiester bonds
GAATTC………..GAATTC
CTTAAG………..CTTAAG
• Cloning vector – original
plasmid used to carry foreign
DNA into a cell and replicate
there
• Bacteria most commonly used
host cells
Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)
• Identification of cells with
clones of the right DNA gene
done by using radioactive
nucleic probes.
• Probes are complements to
part of the gene’s sequence
• DNA must be denatured (Hbonds broken) for probe to
have access to unwound DNA
Figure 20.4 Using a nucleic acid probe to identify a cloned gene
SOME PROBLEMS AND
SOLUTIONS
• Problem: expressing eukaryotic genes
in prokaryotic cells
– Solution: expression vector –
cloning vector that contains the
prokaryotic promoter just
upstream of restriction site and
inserted gene
• Problem: Introns and noncoding
regions in eukaryotes
– Solution: use RNA and reverse
transcriptase to make cDNA
(complementary DNA or cDNA which
has no introns)
• Problem: differences between
eukaryotes and prokaryotes
– Solution: use yeast and some fungi
Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
• Problem: Getting DNA into
eukaryotic cells
– Solutions: Electroporation –
shock to make holes in cell
membrane and
microscopically thin needles
Figure 20.x2 Injecting DNA
PCR
• Polymerase chain reaction –
technique used to quickly copy
(amplify) DNA and without cells
– Used when DNA source is scarce or
impure
– Use heat resistant DNA
polymerase, specific primers, and
nucleotides to make many copies
– Used to amplify a smaller section of
DNA
Figure 20.7 The polymerase chain reaction (PCR)
GEL ELECTROPHORESIS
• Gel electrophoresis – used to separate
macromolecules based upon their size,
charge, and other physical properties
– DNA sample is cut with restriction
enzymes
– Different individuals produce
different sized fragments which move
through gel at different rates
– Different alleles for same gene
produce different sized fragments
GEL ELECTROPHORESIS
• Restriction fragment length
polymorphisms (RFLP) – differences in
DNA sequences on homologous
chromosomes that result in different
restriction fragment patterns
• RFLP’s used as genetic markers for
making linkage maps (distance between
genes)
Figure 20.8 Gel electrophoresis of macromolecules
Figure 20.9 Using restriction fragment patterns to distinguish DNA from different
alleles
Figure 20.9 Using restriction fragment patterns to distinguish DNA from different
alleles
SOUTHERN BLOTTING
• Southern Blotting – hybridization
technique used to determine presence of
specific sequences within DNA
– After gel electrophoresis is done, gel
is blotted with nitrocellulose paper
– DNA is transferred to paper in the
banding pattern
– Paper blot is exposed to radioactively
labeled probes and then rinsed
– Photographic film is laid over paper and
radioactive probes that are attached
to complements expose film
Figure 20.10 Restriction fragment analysis by Southern blotting
Figure 20.17 DNA fingerprints from a murder case
Figure 20.x1a Laboratory worker reviewing DNA band pattern
Figure 20.x1b DNA study in CDC laboratory
DNA SEQUENCING
• DNA sequencing – uses dideoxy
chain termination
– Each copied strand starts with same primer
and ends with a dideoxyribonucleotide
(ddNTP)
– ddNTPs terminate strand because they do
not have 3’ OH
– Each ddNTP is fluorescently labeled
differently (A, T, C, and G)
– Labeled strands are sorted by size in a gel
– Light source reads fluorescence, which
determines base (A, T, C, or G)
DNA sequencing
DNA Microarrays
• DNA microarrays – numerous
amounts of small DNA strands of
known sequences fixed to a glass
slide or chip
• Unknown DNA is labeled with
fluorescent tags
• Labeled DNA is washed over array
and complementary strands bond and
glow
DNA Microarrays
Cloning
• Cloning plants: single-cell cultures
– Works because plant cells can dedifferentiate
• Cloning animals: Nuclear transplantation
– First successful cloned mammal, Dolly, in
1997
– Procedure does not have high success rate
– Often clones have problems
– Probably due to gene regulation differences
in adult and embryo DNA
• Stem cells – relatively unspecialized
cells that can both reproduce itself
independently and, under appropriate
conditions, differentiate into
specialized cells
– Found in embryos, skin, hair, eyes,
dental pulp
• Potentially used to treat many
diseases like Alzheimer’s, Parkinson’s,
Huntington’s etc.
APPLICATIONS OF BIOTECHNOLOGY
• Gene therapy – replacing defective genes with a
normal gene
– In bone marrow cells, embryonic cells, and
gametes
– Problems:
• Even if gene expressed, activity diminishes
• Appropriate amounts expressed/ right time/
right place
• Ex. SCID (severe combined immuno
deficiency) lacks protein
– Attempts to insert gene into bone marrow
cells of 10 children worked in 9 patients,
but caused leukemia in 2 (inserted into cell
division gene)
Figure 20.16 One type of gene therapy procedure
• Pharmaceutical products
– Insulin
– Human growth hormone
– Tissue plasminogen activator (TPA)
• Helps dissolve clots after heart
attack/stroke
– Make genetically engineered proteins to
block or mimic cell receptors
• Experimental drug that mimics
receptor protein that HIV binds to so
it attaches to drug instead of entering
T-cells
• Forensic Science – DNA fingerprinting
• Environmental – using bacteria to clean up toxic
wastes (extract heavy metals)
• Agricultural – vaccines, antibodies, and growth
hormones
– Bovine growth hormone to make produce more
milk
– Genetically modified organisms (GMO)
• Transgenic organisms – organisms that are given
gene(s) from another species
• Plants –genes for resistance to herbicides, less
spoilage, larger fruits etc.
• Medicine – using SNPs (single nucleotide
polymorphisms) as markers; helps to identify
disease causing alleles
Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants
Figure 20.20 “Golden” rice contrasted with ordinary rice
Genes from
daffodils and a
bacterium that is
inserted into rice
plants so that rice
can make betacarotene
Figure 20.18 “Pharm” animals
• Sheep with
gene for
human blood
protein
• Protein is in
sheep milk
and isolated
• Helps inhibit
enzyme that
destroys lungs
in CF patients