Download GENETIC ENGINEERING CHAPTER 20

GENETIC ENGINEERING
CHAPTER 20
• Genetic engineering is the ability of humans to
modify and manipulate DNA for:
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Identification of genetic disorders
Gene therapy
Crop and food production
Tailoring medicines for the cancer
Creating recombinant medicines, vaccines
Forensics
Analyzing evolutionary relationships
• In order to do these, scientists need the genes to
study and sequence
I. DNA Cloning
• Involves inserting DNA into a vector and
cloning it into a cell for production of
multiple copies
A. Vectors
– DNA molecules that will carry foreign DNA into
cells: plasmids, BACs, YACs
• Plasmid: small circular DNA found in bacteria.
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• Antibiotic resistant gene allows selection of bacteria
that took up plasmid
• Multiple cloning site allows insertion of foreign DNA
• Bacterial artificial chromosome (BAC): large
plasmid that can carry large DNA fragments
in bacterial cells
• Yeast artificial chromosome (YAC): derived
from yeast DNA and used to clone really large
DNA fragments into eukaryotic cells
B. restriction enzymes
• found naturally in bacteria to protect them from
invading viruses
• molecular scissors that cut DNA at specific
nucleotide sequences
• enzyme binds to DNA at that sequences and cuts
between the sugar and phosphate on both strands
– cuts can leave blunt or sticky ends
Constructing recombinant DNA:
II. How to clone a gene using a
plasmid vector
A. Basic procedure
– Cut the DNA of interest and vector with the same
restriction enzyme (genomic or cDNA)
– Fragments are mixed and ligase seals fragments
together
– Vector DNA will incorporate the fragments of source
DNA in them creating recombinant plasmid
– Some vectors will NOT incorporate foreign DNA.
They are nonrecombinant
– Cells are transformed by mixing vector with cells
– Recombinant cells are selected for using agar plates
with antibiotics
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B. DNA libraries
• Contain fragmented DNA
from a particular species
stored in a vector ready
for cloning
– Genomic libraries: made
from genomic DNA
– cDNA libraries: made from
a species’ mRNA using
reverse transcriptase
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Constructing a cDNA library:
C. Screening for the gene of interest
• Use a radioactive probe that will only
recognize gene of interest
– Transfer some of the bacteria to a piece of filter
paper forming a replica
– Incubate filter with radioactive piece of DNA
complementary to gene of interest
– Make an X-ray film.
– Use the X-ray film to isolate colony with gene of
interest
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III. DNA technology
A. PCR
• Makes many copies of a
DNA sample
• Uses a three step cycle:
• Heating to about 95o
C to separate strands
• Cooling to anneal
primers
• Replication using ___
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B. Electrophoresis and Blotting
1. Electrophoresis
• Uses a gel as a
molecular sieve to
separate nucleic acids
or proteins based on
size
• A current is applied to
gel that causes the
charged molecules to
move thru the gel
forming bands based
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on size
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• Bands are visualized
with some type of dye
2. Blotting
–After electrophoresis, bands are
transferred to a filter paper for analysis of a
particular fragment (a gene fragment,
mRNA, or protein)
–Filter paper is incubated with a “probe” to
the particular fragment of interest
–Southern blot analyzes DNA
–Northern blot analyzes mRNA
–Western blot analyzes proteins
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Northern blot
3. DNA sequencing
• Small stretches of
DNA are sequenced
using dideoxy chain
termination method
• Uses
dideoxynucleotides
(ddNTP) mixed with
normal ones
• Each type of ddNTP
has a different
fluorescent tag
• DNA sequence is http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch
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Manual sequencing done in 1980s and 1990s
Automated sequencing done now:
IV. The human genome is polymorphic
• Genetically, humans are 99% identical but:
– Alleles have small nucleotide differences
– Different individuals have different numbers of
short tandem repeats: a sequence of 2-5
nucleotides repeated over
– This is restriction fragment length polymorphism
SNPs:
Short Tandem Repeats:
A. Creating genetic profiles using STR
analysis
• There are many regions on chromosomes
where STRs exist.
• The number of tandem repeats in each
region varies from individual
• By using PCR on the STR’s of an individual,
you can create a genetic profile
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Cloning an organism: