DNA Technology What is Genetic Engineering?

fretfulcrunchBiotechnology

Dec 10, 2012 (4 years and 8 months ago)

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DNA Technology
Genetic Engineering
What is Genetic Engineering?
• application
of molecular genetics.
• in other words, it is using or applying what
we know about the DNA molecule and
inheritance to do
stuff.
What kinds of things are we
doing with genetic engineering
technology?
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Genetic Engineering Technology
• 2 Basic Processes:
– Identifying genes
• Disease Diagnosis
• Forensic Testing
• Paternity Testing
• Evolutionary Comparisons
– Gene Transfer
• Gene Therapies
• GM Crops
• Cloning
Identifying Genes
Identifying Genes: DNA Probes
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Identifying Genes: PKU -
phenylketonuria
Identifying Genes: Sickle Cell
Identifying Genes: Cystic Fibrosis
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Identifying Genes: Hemophilia
Identifying Genes: From Probes to
DNA microarrays
Identifying Genes: Forensic Analysis
– RFLP
– PCR
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Identifying Genes:
Evolutionary Relationships
DNA similarities
• Human to Human ~99%
• Human to Chimp ~96-98%
• Human to Mouse ~60%
Organization of
the Human
Genome
HUMAN GENOME
3,300 Mb
~50,000 genes
Genes and gene-related sequences:
25%
Extragenic DNA:
75%
Genes:
40%
(Includes introns)
Gene-related:
60%
Unique or low
copy number:
60%
Moderate to
highly repetitive
40%
Tandem
repetitive:
(a-satellites,
VNTRs, STRs)
Interspersed
repetitive:
(LINEs, SINEs)
Gene
fragments
Pseudogenes
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The tools of
genetic
engineering
1. Restriction Enzymes
Restriction Enzymes
• Proteins that cut DNA at specific sites.
– There are hundreds of different restriction
enzymes available.
– They are isolated from bacterial cells.
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Restriction Enzymes
• The site at which a RE cuts is called a
restriction site.
• Cut locations are determined by specific
nucleotide sequences.
• These sequences are palindromes.
– Was it a rat I saw
– A Man A Plan A Canal Panama
– Dammit, I'm mad
– Madam, I'm Adam
Restriction Enzymes
• DNA Palindromes:
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RE – Sticky Ends
RE – Sticky Ends
• Sticky ends with the same “overhang”
sequence can be used to “glue” DNA
fragments into new combinations.
• If DNA from two organisms is cut with the
same RE ￿new mixed combination can
result
• DNA from more than one organism is
called recombinant DNA.
The tools of
genetic
engineering
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Transplanting Genes
c) Since each RE cuts at a unique nucleotide
sequence ￿each RE will generate a
unique genomic library.
d) With some luck some fragment in one, of
possibly many, genomic libraries will contain
a piece of DNA with the desired gene.
Transplanting Genes
2.Making
recombinant
DNA
• A cloning vector
must be cut
with the same
RE as the
donor gene so
that the
fragments will
combine when
mixed.
Transplanting Genes
3.Transferring the Gene
• The recombinant DNA is then inserted into a
host (transgenic organism)
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How do we make sure that our
“transgenics” really have a
working copy of the gene?
Checking for Success
3.Screening
transgenics for
successful transfers
in the lab saves
time and money.
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Checking for Success
• Remember that
reporter genes are
an important part of
a cloning vector.
• How do we screen
multicellular
organism for
success?
Challenges of Transplanting Genes
Challenges with Transplanting
Genes
• In order to be expressed, the donor gene
must be “on”
– That means that the recombinant DNA must
contain more than just the gene, it has to
have all of the parts that control gene
expression.
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Gene Expression
• Gene Expression is “simple” in
prokaryotes and more complex in
eukaryotes.
– Transplanting human genes into bacteria
requires “engineering” to account for the
differences.
Gene Expression in Prokaryotes
• Promoters/Operators
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Gene Expression in Eukaryotes
• Enhancers
• DNA looping
Gene Expression in Eukaryotes
• Euchromatin
Gene Expression in Eukaryotes
• Methylation
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Gene Expression in Eukaryotes
• mRNA modification
– introns / exons
Gene Expression in Eukaryotes
• mRNA modification
– 5’ cap / 3’ poly A tail
– Control movement out of nucleus &
susceptibility to degradation
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Gene Expression in Eukaryotes
• Movement of mRNA by cytoskeleton
(differentiation)
• Homeotic Genes (Morphogenesis)
• Oncogenes / Proto oncogenes (Cell Cycle)
DNA Fingerprinting
RFLP & PCR
What is a DNA fingerprint?
• A pattern of bands
made by fragments
of an individual’s
DNA.
• We can use it to
establish identity &
paternity.
• We can also use it to
establish
evolutionary
relationships
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Making a DNA fingerprint
RFLP
Restriction
Fragment
Length
Polymorphism
Making a DNA fingerprint
1.Extract DNA from source.
– Sources of DNA:
• Any cell that has it:
• Dandruff (Skin)
• Buccal swab
• Blood (WBC)
• Teeth (blood vessels/nerves)
• Hair (follicle not shaft)
• Etc.
Making a DNA fingerprint
2.Cut DNA w/ RE ￿Genomic Library
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Making a DNA fingerprint
3.Separate
fragments by gel
electropheresis.
What’s gel electrophoresis?
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What’s gel electrophoresis?
• The gel is a matrix
– it has microscopic
holes
– it can sort DNA by size
because small pieces
can move through it
more quickly than big
pieces.
What’s gel electrophoresis?
• Electricity is what
makes the DNA move
through the gel.
• DNA is negatively
charged ￿it moves
from negative to
positive.
What’s gel electrophoresis?
• Big fragments move
slow ￿they don’t
get very far.
• Small fragments
move fast ￿they
travel farther.
• Fragments of the
same size travel at
the same speed.
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Back to how to make a DNA
fingerprint
1.Extract DNA
2.Cut DNA w/ RE
3.Separate with gel electrophoresis
Making a DNA fingerprint
4.Add a probe to help you see the bands of
DNA
• Probes can be florescent or radioactive.
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Making a DNA fingerprint
5.Analyze the print.
Trace Evidence
What if there is only a small sample
of DNA?
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PCR
PCR
1.Heat the DNA to denature. (94)
2.Add DNA primers for both strands & cool
to allow annealing. (54)
3.Add DNA polymerase & Extra
Nucleotides. (72)
4.Repeat.
Taq Polymerase
Thermus aquaticus
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PCR
PCR
• Changing the
temperature is what
could limit our ability
to quickly copy a
strand of DNA.
• Thermocycler
So how are we using PCR for
DNA fingerprinting?
• A standard RFLP analysis uses RE to cut
the DNA and then analyzes the
fragments.
• In PCR we build the allele that we are
looking for.
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So how are we using PCR for
DNA fingerprinting?
• We can specify what part of the genome to
copy during PCR by choosing/using specific
primers.
PCR – What are we looking at?
• VNTRs
– Variable Number Tandem Repeats
• FBI CODIS Database
– 13 (15) VNTR markers
Organization of
the Human
Genome
HUMAN GENOME
3,300 Mb
~50,000 genes
Genes and gene-related sequences:
25%
Extragenic DNA:
75%
Genes:
40%
(Includes introns)
Gene-related:
60%
Unique or low
copy number:
60%
Moderate to
highly repetitive
40%
Tandem
repetitive:
(a-satellites,
VNTRs, STRs)
Interspersed
repetitive:
(LINEs, SINEs)
Gene
fragments
Pseudogenes
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Characteristics of Extragenic DNA
• Most of it has no known function
– Sometimes called “junk DNA”
• Mutations do not affect phenotype
– Mutations tend to accumulate over time
– Rich source of DNA polymorphism
• Tandemly repeated regions are “mutation hot spots”
– Strand slippage mis-pairing during DNA replication
Strachan and Read
(2004)
What Makes a “Good”
Human DNA Marker?
• Everyone has the marker
• Highly polymorphic (many possible alleles)
• Fairly equal allele frequency distributions
• Small enough to be assayed by PCR
– Allows trace evidence to be analyzed
– Allows degraded evidence to be analyzed
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D7S280
• aatttttgta ttttttttag agacggggtt
tcaccatgtt ggtcaggctg actatggagt
tattttaagg ttaatatata taaagggtat
gatagaacac ttgtcatagt ttagaacgaa
ctaac
gatag atagatagat agatagatag
atagatagat agatagatag atagatagat
tgata
gtttt tttttatctc actaaatagt
ctatagtaaa catttaatta ccaatatttg
gtgcaattct gtcaatgagg ataaatgtgg
aatcgttata attcttaaga atatatattc
cctctgagtt tttgatacct cagattttaa
• Common alleles: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
AmpFlSTR Identifiler™ Allelic Ladder
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Genotyping with STRs:
• Steps:
– Extract DNA from source sample(s)
– Amplify the VNTRs by the polymerase
chain reaction (PCR)
– Separate and detect amplified alleles by
capillary gel electrophoresis (CGE)
Primer Location on a typical STR
7 repeats
8 repeats
AATG
PCR primers
Labeling of Primers
• For PCR amplification of VNTRs, the primers are
labeled with fluorescent dyes
• 4-5 different dyes are utilized
– Dyes fluoresce at different wavelengths
– If 15 STRs are amplified, a 5-dye system is used and all
15 STRs are amplified in one PCR reaction (multiplex
PCR)
– If 13 are used, two multiplex reactions with 4 dyes
• Profiler (9 STR + amelogenin)
• Cofiler (6 STR + amelogenin; two overlap with Profiler)
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Separation and
Detection
PCR sample
loaded into
capillary
Samples run through
capillary according to size
(+)
(-)
As PCR products pass
capillary window, a laser
excites the fluorescent
tag and the tag emits a
signal
The signal is sent
to a computer for
interpretation and
analysis
CCD
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Locus Mother's Alleles Child
Obligate
Paternal Allele AF Alleles
D3S1358 14 15 15 16 16 16 17
vWA 14 16 14 16 NI 14 16
FGA 21 24 21 22 22 22 22
D8S1179 12 13 12 12 12 13 14
D21S11 29 33.2 30 33.2 30 29 31
D18S51 15 21 15 18 18 12 15
D5S818 12 12 12 13 13 11 11
D13S317 11 12 12 13 13 11 13
D7S820 9 11 10 11 10 11 11
D16S539 11 11 9 11 9 9 9
THO1 7 9.3 7 9.3 NI 7 7
TPOX 6 8 6 8 NI 7 7
CSF1PO 7 12 11 12 11 11 11
Amel X X X Y Y X Y
Locus Mother's Alleles Child
Obligate
Paternal
Allele AF Alleles
D3S1358 17 18 15 18 15 15 16
vWA 15 19 16 19 16 16 19
FGA 22 22 22 25 25 23 25
D8S1179 13 15 15 15 15 14 15
D21S11 28 30.2 28 30 30 30 30.2
D18S51 15 15 13 15 13 13 13
D5S818 10 12 12 12 12 12 12
D13S317 11 12 12 12 12 12 12
D7S820 9 10 9 10 NI 8 10
D16S539 9 10 10 10 10 10 11
THO1 6 7 6 9.3 9.3 7 9.3
TPOX 6 8 6 8 NI 6 8
CSF1PO 11 12 11 11 11 10 11
D2S1338 22 24 24 24 24 17 24
D19S433 10 13 10 13 NI 13 16
Amel X X X X X X Y
Paternity Index (PI)
• PIs are calculated for each of the 15 loci
these are then multiplied together to give a
Combined Paternity Index (CPI) for the test
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Sample PI Calculation
• The obligate paternal allele is “15” at D3S1358
• The alleged father is African American
• The alleged father’s genotype is (15,16) so his probability of
passing the allele to a child is 0.5
• The frequency of the “15” allele is 0.2795 in the African
American (AA) population.
• PI = 0.5/0.2795 = 1.79
• Thus, it is 1.79 times more likely
that the alleged father could
pass the obligate paternal allele at this locus to the child than
a random AA man
Sample PI Calculation
• If the alleged father had been a
homozygote for the “15” allele, the PI
calculation would have been:
(1)/.2795 = 3.58
• Therefore, it is now 3.58 times more likely
that the alleged father could pass the
obligate paternal allele to a child than a
random man could do so.
Combined Paternity Index
Locus
Mother's
Alleles Child
Obligate
Paternal
Allele AF Alleles
Freq AF
Allele PI
D3S1358 17 18 15 18 15 15 16 0.2795 1.79
vWA 15 19 16 19 16 16 19 0.2692 1.86
FGA 22 22 22 25 25 23 25 0.1231 4.06
D8S1179 13 15 15 15 15 14 15 0.2103 2.38
D21S11 28 30.2 28 30 30 30 30.2 0.1615 3.10
D18S51 15 15 13 15 13 13 13 0.0410 24.39
D5S818 10 12 12 12 12 12 12 0.3256 3.07
D13S317 11 12 12 12 12 12 12 0.4436 2.25
D7S820 9 10 9 10 NI 8 10
D16S539 9 10 10 10 10 10 11 0.1128 4.43
THO1 6 7 6 9.3 9.3 7 9.3 0.1000 5.00
TPOX 6 8 6 8 NI 6 8
CSF1PO 11 12 11 11 11 10 11 0.2128 2.35
D2S1338 22 24 24 24 24 17 24 0.0800 6.25
D19S433 10 13 10 13 NI 13 16
Amel X X X X X X Y
CPI 5,459,751
Thus, it is 5,459,751
times more likely that the alleged
father could have contributed the obligate paternal alleles to
the child than a random man. A CPI of 100 is sufficient to
establish paternity in a court of law
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Probability of Paternity
• In a Probability of Paternity calculation, the prior probability of
paternity is assumed to be 0.5
• This means that we are assuming, before any genetic testing, that
the alleged father and the child’s mother have an equal chance of
telling the truth
• Under these conditions: PP = CPI/(CPI+1)
• Thus, if CPI = 5,459,751 (as in prior example):
• PP = (5,459,751/(5,459,751+1) = 0.999999817
Paternity: Special Cases
• Single Exclusions
– VNTRs are mutation hot spots
– The average rate of germ-line mutation per Identifiler

locus is
.00136 (1 per 735 sperm)
– With 15 loci, ~1 in 50 sperm is likely to carry at least one germ-line
mutation at one of the 15 loci
– Single exclusions are common in paternity labs and an exclusion
of an alleged father is never made on the basis of a single locus
– PI is calculated using known mutation rate at locus
Heteropaternal Superfecundation
• Fraternal twins with different fathers
– Occasionally, genetic tests exclude the alleged father
for one twin and include him for the other twin
– Requires that the mother ovulated twice during her
cycle and had sexual relations with two different men
while fertile
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But it’s not always easy to tell!
• Fraternal twins with
the same father
• Both parents mixed
ethnicity: 50%
African-American,
50% Caucasian
• The “Talk Show Effect”
– In the lab, testing 2-3 different alleged fathers before
finding an inclusion is not uncommon
– The most men tested in a nearby lab for a single
paternity test was 8
– Rates of non-paternity in the general population run
at about 10%
• True across all U.S. ethnic groups
• True across most developed nations