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Manipulating DNA
Manipulating DNA
• Scientists use their knowledge of the structure of
DNA and its chemical properties to study and
change DNA molecules
• Different techniques are used to study and
change DNA molecules
• Genetic Engineering – making changes in the
DNA code of a living organism
• Bacteria are the workhorses of modern
biotechnology
Tools of Molecular Biology:
Recombinant DNA
• Recombinant DNA techniques can help biologists
produce large quantities of a desired protein
• To work with genes in the laboratory, biologists often use
bacterial plasmids, small, circular DNA molecules
– Plasmids can:
• can carry virtually any gene,
• can act as vectors, DNA carriers that move genes from one
cell to another, and
• are ideal for gene cloning, the production of multiple identical
copies of a gene-carrying piece of DNA.
Recombinant DNA
• Recombinant DNA is produced by
combining two ingredients:
• a bacterial plasmid and
• the gene of interest.
• To combine these ingredients, a
piece of DNA must be spliced into a
plasmid
Creating Recombinant DNA
• In order to create Recombinant DNA, there needs to be:
– DNA extraction
• Cells opened to separate DNA from other cell parts
– Cutting DNA
• DNA too large to study, so biologists “cut” them into smaller
fragments using restriction enzymes. Many restriction enzymes
are known and each one cuts DNA at a specific sequence of
nucleotides
• Produces pieces of DNA called restriction fragments with “sticky
ends” important for joining DNA from different sources.
– Splicing DNA back together
• DNA ligase connects the DNA pieces into continuous strands by
forming bonds between adjacent nucleotides
Recognition site (recognition sequence)
for a restriction enzyme
Restriction Enzymes
1 A restriction enzyme cuts the
DNA into fragments.
Restriction
enzyme
Recognition site (recognition sequence)
for a restriction enzyme
DNA
Restriction Enzymes
1 A restriction enzyme cuts the
DNA into fragments.
2
A DNA fragment is added from
another source.
Restriction
enzyme
Recognition site (recognition sequence)
for a restriction enzyme
DNA
Restriction Enzymes
1 A restriction enzyme cuts the
DNA into fragments.
2
A DNA fragment is added from
another source.
3 Fragments stick together by
base pairing.
Restriction
enzyme
Recognition site (recognition sequence)
for a restriction enzyme
DNA
Restriction Enzymes
1 A restriction enzyme cuts the
DNA into fragments.
2
Restriction
enzyme
A DNA fragment is added from
another source.
3 Fragments stick together by
base pairing.
4
DNA ligase joins the fragments
into strands.
DNA
ligase
Recombinant DNA molecule
Restriction Enzymes
Recognition sequences
DNA sequence
Restriction enzyme
EcoRI cuts the DNA
into fragments.
Sticky end
Restriction Enzymes
Recognition sequences
DNA sequence
Restriction enzyme
EcoRI cuts the DNA
into fragments.
Sticky end
Finding the Gene of Interest
• How can a researcher obtain DNA that encodes
a particular gene of interest? First, you have to
have an idea of what the gene is you want to
work with (get a genomic library). Then:
• Using a nucleic acid probe consisting of a short
single strand of DNA with a complementary
sequence and labeled with either a radioactive
isotope or a fluorescent dye.
• Or by synthesizing it through reverse transcriptase
(viral enzyme that makes DNA)
• Or by making it by scratch with machines
DNA Profiling and Forensic
Science
• DNA profiling
• can be used to determine if two samples of genetic
material are from a particular individual and
• has rapidly revolutionized the field of forensics, the
scientific analysis of evidence from crime scenes.
– To produce a DNA profile, scientists compare
sequences in the genome that vary from
person to person.
DNA Profiling and Forensic
Science
– DNA profiling can be used to
•
•
•
•
•
test the guilt of suspected criminals,
identify tissue samples of victims,
resolve paternity cases,
identify contraband animal products, and
trace the evolutionary history of organisms.
DNA Profiling Techniques:
Making Copies of DNA
• Polymerase Chain Reaction (PCR)
technique
– Allows biologists to make many copies of a
specific piece of DNA
– DNA strands separated with heat, then cooled
to allow DNA Polymerase to start making new
copies of DNA
– A few dozen heat and cool cycles results in
many copies of DNA
Making Copies of DNA
DNA polymerase adds
complementary strand
DNA heated to
separate strands
DNA fragment
to be copied
PCR
cycles 1
2
3
4
5 etc.
DNA
copies 1
2
4
8
16 etc.
DNA Profiling Techniques: STR
Analysis
– Short tandem repeats (STRs) are:
• short sequences of DNA that are repeated many times,
tandemly (one after another), in the genome.
– STR analysis
• Proves two samples come from the same person - Everyone
has these repetitive DNA sequences, but in different lengths
and a different number of them
• compares the lengths of STR sequences at specific sites in
the genome
• uses gel electrophoresis, a method for sorting DNA by size
DNA Profiling Techniques: Gel
Electrophoresis
• Gel electrophoresis
– Used to separate DNA fragments. DNA
fragments placed in a gel and electricity is
applied to the gel. DNA molecules are
negatively charged and move towards the
positive end of the gel. Smaller DNA
fragments move faster and farther
– This technique used to compare the genomes
of different organisms or even different people
Gel Electrophoresis
Power
source
DNA plus restriction
enzyme
Longer
fragments
Shorter
fragments
Mixture of DNA
fragments
Gel
Gel Electrophoresis
Gel Electrophoresis
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