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Ch. 19
Part 1
Gene Technology
Genetic Engineering (GE)
• modification of an organism's genetic composition
by artificial means
• involves transfer of genes from one organism into a
plant or animal of an entirely different species
• Uses of GE:
– understanding that changes in DNA  changes in
protein synthesis
– Sequencing nucleotides in DNA & comparing them
to different organisms
– Carry out genetic tests to determine occurance of
disorders
– Gene therapy to treat diseases
• Goal:
– Remove gene(s) from one organisms & transfer to
another organism so gene is expressed in the new
organism
• Issues:
Selective Breeding
• Involves whole sets of genes
Genetic Engineering
• Involves transfer of a single
gene from one organism to
another
Recombinant DNA (rDNA)
• DNA that has been
altered by genetic
engineering
• Made by joining DNA
from 2 different sources
• Contains lengths of
nucleotides from
different organisms
• Organisms containing
rDNA are called
TRANSGENIC
ORGANSIMS
– Aka genetically modified
organism GMO)
– Definition: organism
containing recombinant
DNA that expresses new
genes
Transgenic Organisms
Steps of Gene Transfer
1. Identify gene
–
Make desired gene using one of
these methods:
•
•
•
Gene cut from chromosome
Reverse transcription of mRNA
Artificial synthesis from nucleotides
2. Make copies of gene
–
Use Polymerase Chain reaction
(PCR)
3. Insert gene into vector
–
Vectors deliver gene into the cells
of organisms
Examples:
–
•
•
•
Plasmids
Viruses
liposomes
4. Vector carries gene into cells
5. Cells with new gene identified
and clones
3 Main Tools for a Gene Technologist:
• Enzymes
– Restriction nucleases
– Ligases
– Reverse transcriptase
• Vectors
– Plasmids
– Viruses
– liposomes
• Genetic Markers
– Genes coding for easily
identifiable markers that can be
used as markers
Bacteriophages
• Virus
• Infects bacteria
• “phages”
Tools for a Gene Technologist:
Restriction Enzymes (RE)
• Restriction endonucleases
– Class of enzymes from bacteria that breakdown DNA of invading
virus (bacteriophage)
– Bacteria contain gene that codes for enzymes that cut the phage
DNA at sugar-phosphate backbone into smaller pieces at specific
spots of the DNA
– Purpose: to restrict viral infection of bacteria (protect bacteria)
How Restriction Enzymes Work
• Bind to specific TARGET SITE of
DNA
– Called the Restriction site: a specific
sequence of bases
– Bacterial DNA is NOT cut by enzyme
because:
• Protective chemical markers OR
• Does not have target/restriction site in
its DNA
– Usually a PALINDROMIC nucleotide
sequence
• Reads the same in both directions
• RACE CAR (same both ways)
• GGATCC and complementary strand is
CCTAGG
How Restriction Enzymes Work
• TWO Ways to cut viral DNA:
– Staggered patter across sugarphosphate backbone  STICKY
ENDS
• short region of UNPAIRED nucleotides
• Unpaired region is called an overhang
– sticky because it wants to and will pair
with another sticky end that has
complementary overhang sequence
– Sticky ends are like long-lost twins
seeking to hug each other tightly once
they meet
• EASY for hydrogen bonds to form with
complementary bases with another
piece of DNA cut with same restriction
enzyme
– Straight across sugar-phosphate
backbone  BLUNT ENDS
• not sticky because all the nucleotides
are already paired between the two
strands of DNA
• blunt ends of DNA and plasmids are
less likely to find each other, and thus
ligation of blunt ends requires that
more DNA is put into the test tube
What happens when you cut LONG pieces of DNA with RE?
• DNA pieces of
different lengths are
created
• Need to separate
DNA pieces by length
in order to locate a
specific piece
– Use GEL
ELCTROPHORESIS
and GENE PROBES
• Once you have
desired DNA
segment, you make
millions of copies of
that piece using
POLYMERASE CHAIN
REACTION (PCR)
Naming Restriction Enzymes
• Abbreviation indicates ORIGIN of restriction enzyme
– Organism it came from
• Roman numerals  differentiate between enzymes
from the same source
– Usually numbered by order which they were
found/discovered
• EcoRI
– From Escherichi coli bacteria (Strain RY)
– “I” means it was the first restriction enzyme made from E.
Coli
Improvements in Gene Technology
• Protein sequencing:
– technique to determine the
amino acid sequence of a
protein, as well as which
conformation the
protein adopts
– Genetic code used to
determine order of
nucleotides & synthesize DNA
artificially from nucleotides
• Better than cutting gene from
chromosome or using reverse
transcriptase
Improvements in Gene Technology
• DNA templates no longer needed to make genes
or genome
• Steps to create new “novel” genes (such as those
in a vaccine)
– Choose codons for desired amino acid sequence
– Sequence of nucleotides held in computer
– This directs synthesis of short fragments of DNA
– Fragments of DNA joined to make longer sequences
of DNA
– Longer sequences of DNA inserted into plasmids for
use in genetic engineering