Download DNA Technology

DNA Technology
G/T Biology I
Uses of DNA Technology



Purposely mutate genes to find out what they do
(must mutate developing embryo)
Clone genes (put human genes into bacteria makes a lot of copies of a gene to study it,
sequence it, or make large amounts of a protein)
See if an embryo has a defective gene (use
complementary DNA pieces to “tag” a gene)

Make vaccines (make large amounts of
proteins that trigger the immune response)

Gene Therapy (putting genes into somatic or
germ cells)
Uses of DNA Technology Involving
Gel Electrophoresis
 Identify
recessive alleles
Diagnosis of disease
 Study of relatedness of species
 Crime Solving (DNA fingerprinting)
 Paternity Testing
 Figuring out the sequence of a gene

Restriction Enzymes – What are
they and why do you need them?


They are enzymes that cut DNA at very specific
sequences
We need them for any kind of recombinant DNA
technology (genetic engineering) – splicing
genes into different DNA


Ex. Putting the human insulin gene into bacteria so
that we can get bacteria that multiply quickly to make
buckets of human insulin for diabetics
We need them for any DNA technology involving
gel electrophoresis
Enzyme
Organism from which derived
(cut at *)
5' -->3'
Ava I
Anabaena variabilis
C* C/T C G A/G G
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
GG*CC
Hha I
Haemophilus haemolyticus
GCG*C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * AA C
Kpn I
Klebsiella pneumoniae
G GTAC * C
Mbo I
Moraxella bovis
*G A T C
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
SstI
Streptomyces stanford
GAGCT*C
Sal I
Streptomyces albus G
G*TCGAC
Taq I
Thermophilus aquaticus
T*CGA
What is Electrophoresis?
o
o
o
o
Electrophoresis is the separation of molecules
(pieces of DNA) in a porous matrix based on
electrical charge and size
The porous matrix is a gel
If you put an electrical charge through the gel
material and put the DNA at the negative pole, it
will migrate to the positive pole since DNA is
negatively charged
If you cut the DNA into pieces and then run it
thru the gel, the bigger pieces of DNA will move
slower, the smaller pieces will move faster –
therefore the pieces of DNA will separate
How do we use Electrophoresis to Diagnose a
Disease or Identify a Recessive Allele
Cut the DNA (if you have purified the gene)
 Run gel (DNA runs toward the + pole)
 Stain gel
CCG↓CGGTAGGAAC CCACGGTAGGAAC
____
____

____
How do you use Electrophoresis for
DNA Fingerprinting?






Even though human DNA is 99.9% the same, 0.1% is 6
million differences in individual nucleotides.
Through research, we have identified regions of the DNA
that is the most variable and cut those areas with
restriction enzymes
This will make different sized fragments based on the
enzymes cutting the DNA differently
If we run them on a gel, we will see different patterns
The same person always has the same pattern because
the DNA doesn’t change
Different people may have the same pattern if they have
the same mutations in the areas of the DNA analyzed
This is an example of
a real DNA fingerprint
from a real case
How to Use Electrophoresis for
Paternity Testing



Cut the DNA up with
restriction enzymes and
run it on a gel
Every allele the child has,
must come from the
mother or father
If you know who the
mother is, any band that
didn’t come from her, must
be from the father
PCR – polymerase chain reaction






Make millions of copies of a single piece of DNA
(like DNA replication in a test tube)
DNA can be old and in very small quantities
Can use for crime detection if only have 1 cell or
a small sample
Basically, throw in DNA into a test tube with
nucleotides, DNA polymerase
Heat it up to separate the DNA and cool it down
for it base pair back together
The DNA polymerase is special – isolated from
bacteria that live at 160 degree water
50º
A. Double
strand DNA
96º
B. Denature
50º
C. Anneal
primers
Taq
D. Polymerase
binds
72º
Taq
72º
Taq
Taq
Taq
E. Copy
strands
Taq
1
96º
2
3
4
F.
Denature