Ch 20 Gene Technology

Ch 20 Gene Technology

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DNA Identification

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0.10% of DNA varies
btwn each human,
except identical twins

•
Identification compares
samples in regions of a
chromosome that differ

•
Very useful

–
Paternity, human
remains, human origins,
evidence

Noncoding DNA

•
Noncoding DNA
-

98%
does not code for
proteins

–
Contains length
polymorphisms
–

variations in length of
DNA molecule btwn
known genes

–
Some short, repeating
sequences
-

VNTR


VNTR

•
Variable Number Tandem
Repeat

–
Repeat a few or many
times in tandem

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Occurs at specific loci

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# varies among individuals

–
VNTR frequency calculated
in general pop.

–
Useful in determining how
rare a particular DNA
profile is

Copying DNA: Polymerase Chain
Reaction (PCR)

•
PCR
–

technique that quickly
produces many copies of a
DNA fragment

•
Requires

–
template (sequence to be
copied)

–
Supply of 4 DNA nucleotides

–
Heat tolerant polymerase

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Primers
–

artificially made
pieces of single
-
stranded DNA
that are 20
-
30 nucleotides long

•
Must be present for DNA
polymerase to initiate
replication

•
Complimentary to ends of DNA
fragment to be copied


Copying DNA: Polymerase Chain
Reaction (PCR)

1.
Primers bind to DNA

2.
DNA polymerase makes
copy of both strands

3.
Heating breaks bonds
holding template DNA
to newly made strands

4.
After cooling, primers
can bind again to DNA

5.
DNA polymerase can
copy again

6.
Cycle
is repeated


Cutting DNA: Restriction Enzymes

•
Restriction Enzymes

–
Bacterial proteins used
for cutting long DNA
molecules into shorter
pieces

–
Recognize specific short
DNA sequences & Cut in
or near it

–
DNA overhangs are left
& act as sticky ends so
other pieces of DNA can
connect

Sorting DNA by Size: Gel Electrophoresis

•
Separates nucleic acids
(NA) or proteins according
to size

•
Steps:

1.
DNA samples are cut
w/restriction enzyme &
placed in wells made on
thick gel

2.
Electric current runs thru
gel .
-

Charged fragments
migrate toward + end.
Smaller fragments migrate
faster & farther than longer,
separating by size


3.
DNA is transferred to
nylon membrane &
radioactive NA probes are
added. Probes bind to
complementary DNA

4.
X
-
ray film is exposed to
radio
-
labeled membrane

5.
DNA fingerprint
–

resulting pattern of bands

Accuracy of DNA fingerprints

•
Combined analysis of
many VNTR loci

•
1 loci ~ 1 digit of phone
#

•
5
–

15 loci (usually 13)
produce 1 in 100 billion
odds that 2 people will
share DNA profile.

•
6.5 Bil. people on earth

Recombinant DNA
–

Genetic Engineering

•
Altering genetic
material of cells or
organisms to make new
substances

•
Blood vessel growth in
Zebrafish with GFP
–

green fluorescent
protein


Cloning vectors

•
Clone
–

exact copy of
DNA segment, whole
cell or complete
organism

•
Insert vectors into
bacterium or yeast

•
Cloning vectors incl.
viruses that infect
bacteria & plasmids

Plasmids

•
Small rings of DNA
found naturally in some
bacterial cells in
addition to main chrom.

•
Restriction enzyme
used

•
Gene for insulin
inserted

Probes

•
Strand of RNA or 1
-
stranded DNA labeled
with radioactive element
or fluorescent dye base
pairs w/specific DNA

•
Determine which of
1000s of colonies contain
desired gene

•
Xfer

gene from DNA to
filter paper & view under
UV light

Human Genome Project (HGP)

•
Linked >20 scientific
laboratories in 6 countries.

•
3.3
bil
. Nucleotides

•
Every gene on each
chrom
.
was mapped

•
≠ distribution of
exons

(sequences that are
xlated
)

•
Smaller than prev.
estimated

•
SNP
–

single nucleotide
polymorphisms