Download Genetic Technology

GENETIC TECHNOLOGY
1) GENETIC CLONING OVERVIEW
1.
2.
3.
4.
Remove bacterial plasmid with
restriction enzymes
Add in gene of interest (plasmid
is now recombinant DNA
molecule)
Put back into bacteria
Many reproductive cycles later
= amplification of gene &
protein it makes
RESTRICTION ENZYMES
 Cut
up foreign DNA
 Very specific
 Recognize short nucleotide
sequences  cut at specific points
within sequence
 Bacteria’s own DNA is methylated
to protect itself
 What
could we use this for????
RESTRICTION ENZYMES
 Enzyme
finds specific recognition
sequence (restriction site)
 Same sequence found on both
strands, running antiparallel
 Enzyme cuts phosphodiester bonds
of strands
 These restriction fragments are
double stranded with single
stranded ends (“sticky ends”)
RESTRICTION ENZYMES
 Single
strands will
hydrogen bond with
other complementary
“sticky ends”
 Bonds made permanent
with DNA ligase
 Now we have
recombinant DNA
FROM RESTRICTION ENZYMES
TO PLASMID MAPS
 PM
show how different REs act
upon a plasmid
 Pictorial representation of the
different lengths
of pieces
remaining
after the
REs worked
FROM RESTRICTION ENZYMES
TO PLASMID MAPS
 Procedure
 Think
of plasmid as
clock – from 12 to 12 =
total # base pairs
 Approximate location
of cut based on base pair
fragment length
 Use logic to solve
 Double check based on data
GENETIC RECOMBINATION
 Occurs
with help of plasmids
(cloning vector)
 DNA molecule that can carry
foreign DNA into a cell &
replicate there
2) POLYMERASE CHAIN REACTION
(PCR)
 Can
quickly amplify specific DNA
without using cells
 DNA of interest incubated with
DNA polymerase, nucleotides, &
ss primer DNA for synthesis
 DNA heated  strands separate
 Cool  primers bond
 DNA polymerase adds to 3’ end
of each primer
 Repeat
3A) DNA ANALYSIS (GE)
 Sequence
of entire genome 
genomics
 Begins with gel electorphoresis
 Sorts DNA based on size &
charge
 Can combine with specific
probes to
label
particular
DNA
bands
3B) DNA ANALYSIS (SB)
 Can
also use Southern blotting
 Helps to detect restriction
fragment length polymorphisms
(RFLP)
Differences in DNA sequences
on homologous chromosomes
Can result in different patterns
of restriction fragment lengths
Genetic marker for making
linkage maps
Led to Human Genome Project
4) GENOME ANALYSIS
 Scan
sequence for start & stop
codes, RNA splicing sites, known
genes
 Found 30,000-40,000 genes
 Can figure out new genes by
comparing to old genes with
similar sequence
4) GENOME ANALYSIS
 DNA
microarray:
ssDNA fragments
fixed to slide that
are then labeled
with fluorescent
cDNA
 Compare genes of
species  attempt to
uncover gene function
5) GENOME ANALYSIS - GENE
FUNCTION
 To
determine, turn gene off – see
what happens
 To turn off: RNA interference
(RNAi)
 Synthetic ds RNA matches gene
sequence – binds to mRNA
 Triggers breakdown of mRNA 
no protein made
 Remove to turn on again
THE FUTURE
 Proteomics:
study of full protein
sets
 Study of variations among the
species
 Form of single nucleotide
polymorphisms (SNPs)
Single base-pair variations
One per 1000 bp
DNA TECHNOLOGY
APPLICATIONS
 Disease
Diagnosis
 Use PCR & labeled nucleic acid
probes to detect pathogens (ex:
HIV)
 Identification of harmful
alleles before birth
DNA TECHNOLOGY
APPLICATIONS
 Human
Gene Therapy
 Alteration of genes
 Replace defective gene with
normal one  put into cells
that keep dividing
Appears to be temporary
 Raises ethical questions
DNA TECHNOLOGY
APPLICATIONS
 Pharmaceutical
Products
 Use vector DNA to create
human insulin, HGH, TPA, etc
 Recombinant DNA to make
vaccine without using actual
pathogen
DNA TECHNOLOGY
APPLICATIONS
 Forensics
 Microsatellite
DNA highly
variable between individuals
Called simple tandem repeats
(STR)
 Environmental
 Genetically engineered microbes
to degrade toxic waste
DNA TECHNOLOGY
APPLICATIONS
 Agriculture
 Transgenic
organisms: carry
genes from another species
Makes “super” species
Remove egg  fertilize in vitro
 inject desired DNA into egg
nuclei  cell will grow &
express gene  egg put into
surrogate
DNA TECHNOLOGY
APPLICATIONS
 Plants
Vector is recombinant Ti
plasmid – inserts into plant
genome  cell grows into
complete plant
Can increase
nutritional
value
