7.1 techniques for producing and analyzing dna
TRANSCRIPT
SBI4U
Ms. Ho-Lau
7.1 Techniques for Producing and Analyzing DNA
What is Biotechnology?
From Merriam-Webster:
“the manipulation of living organisms or their components to produce useful usually commercial products (as pest resistant crops, new bacterial strains, or novel pharmaceuticals)”
Biotechnology
Molecular Biology
study of the structures and functions of
nucleic acids and proteins
can be done outside of the cell, in the
controlled environment of a test tube.
Techniques for Producing and Analyzing
DNA
A. Recombinant DNA Technology
B. Gene Cloning
C. Polymerase Chain Reaction (PCR)
D. Analyzing DNA Fragment Size
E. DNA Fingerprinting
F. DNA Sequencing (Dideoxy Sequencing)
G. Making Sequence-Specific Mutations
A. Recombinant DNA Technology
Recombinant DNA: a molecule of DNA composed of genetic material from different sources.
Prokaryotes have many different restriction enzymes that enable them to protect themselves against viral DNA. Restriction enzymes cleave viral DNA so that it can no longer replicate within the organism.
A special type of restriction enzyme is called restriction endonuclease
Restriction Endonuclease
Each restriction endonuclease recognizes a specific nucleotide sequence (target sequence)
The enzyme cuts the double stranded DNA at the restriction site of the target sequence.
G/AATTC
Palindromic sequence
Restriction Endonuclease - Characteristics
Sequence specificity:
Cuts made by restriction endonucleases are specific and predictable
produce identical sets of DNA fragments called restriction fragments.
Staggered cuts:
Most restriction endonucleases produce a staggered cut that leaves regions, called sticky ends, at the ends of fragments.
Sticky ends can form base pairs with other single-stranded regions that have a complementary sequence.
Restriction Endonuclease - Characteristics
Some restriction endonucleases produce blunt ends.
While this allows any two DNA fragments with blunt ends to
combine, many useless by-products form due to lack of specificity.
Restriction Endonuclease – use in bacteria
By adding methyl to the
recognition sites of
endonucleases, the enzyme will
not destroy its own DNA.
Bacteriophage DNA does not
contain methyl groups and thus
is destroyed.
Recombinant DNA Technology
Use restriction enzyme to insert DNA of interest into a plasmid
The sticky ends of the DNA can
combine to any other DNA that
also have complementary sticky
ends (both DNA strands are cut
with the same restriction enzyme)
When both pieces of DNA bind
together it is known as
‘Recombinant DNA’.
Step 1: Restriction enzyme is used to cleave
the DNA of interest (eg. human growth
hormone)
Step 2: the DNA of interest is now
separated as DNA fragment with sticky
ends
Step 3: The DNA from another source (eg.
plasmid) must be cut with the same
enzyme to produce complimentary sticky
ends
Step 4: Both DNA fragments are incubated
with DNA ligase so that a covalent bond
can be formed between both fragments.
B. Gene Cloning
With Recombinant DNA technology, scientists can produce many identical copies of a gene in a process called gene cloning
to study specific genes and proteins
Bacteria are often used as host systems for gene cloning (inexpensive to maintain and can grow easily in large amounts)
Gene Cloning in Bacteria
Recombinant DNA: the gene
of interest is carried in a
vector
Vector: is a self-replicating,
circular piece of DNA called
a plasmid
Gene Cloning in Bacteria
The plasmid must have:
1. its own origin of replication
2. selectable markers (antibiotic resistance
gene and lacZ gene)
3. restriction sites that allow gene of
interest to insert
Gene Cloning in Bacteria
The lacZ gene codes for an enzyme that
breaks down galactose.
The gene of interest is inserted into the
plasmid so that it disrupts the lacZ gene
to make it inactive (and preserve the
galactose).
Gene Cloning in Bacteria
DNA is taken up by the bacteria through transformation
chemicals are used to make the cell membrane more permeable
bacterial cells are plated on a Petri dish containing growth media
supplemented with:
1. antibiotic ampicillin
2. a galactose derivative, X-gal
(causes bacterial colonies to
turn blue when the lacZ gene is
active)
Gene Cloning in Bacteria
bacterial colonies containing the recombinant DNA are identified
using selectable markers
Blue colonies have an active lacZ gene and
contain plasmid only.
White colonies contain the recombinant DNA
of interest since they have an inactive lacZ
gene.
white colonies are transferred to a liquid
culture to grow in larger quantities.
Summary of Gene Cloning
C. Polymerase Chain Reaction (PCR)
invented by Kary Mullis
To produce a large amounts of DNA (DNA
amplification)
does not require a host system or recombinant
DNA construction
an automated process
amplifies specific regions of DNA
from small quantities of template DNA, billions
of copies of DNA can be made in a few hours
Steps in PCR
Step 1: double stranded DNA is denatured in high temperature (95ᴼC).
Step 2: The temperature is lowered (55ᴼC) so that DNA primers can anneal to the 3’ end of both DNA strands.
Steps in PCR
Step 3: Temperature is increased to
72ᴼC and Taq polymerase is added
to the sample. Taq polymerase adds
free nucleotides to the primers that
are complementary to the template
DNA strand.
Step 4: The steps 1-3 are repeated multiple
times (30 – 40 cycles). Each round of
replication generates two new DNA strands.
DNA is amplified exponentially.
D. Gel Electrophoresis
a common method used to analyze,
identify, and purify DNA fragments
1. DNA is digested by restriction enzyme
(broken into DNA fragments)
2. DNA is then loaded into preformed
wells in the gel
3. uses an electric field to separate DNA
fragments based on their mass and
charge
Properties of DNA
1) DNA is negatively charged due to the
phosphate group on the backbone
2) The molar mass of each nucleotide is
approximately the same
THUS, each nucleotide has the same
charge-to-mass ratio
The only difference between fragments is
the length of the fragment
Analyzing DNA Fragment Size
A buffer solution such as agarose or a polyacrylamide gel is added
to the box. The buffer creates pores of different diameters.
A dye is also added to the DNA fragment solution so that it can be
visible when added to the buffer solution.
The smaller DNA fragments have
smaller molecular mass and travel
through the pores of the gel much
easier than larger fragments.
** DNA is treated
with chemicals
that make it
visible
Running the Gel
E. DNA Fingerprinting
analyzes the DNA sequence of certain regions of a person’s
genome
Restriction enzymes and gel electrophoresis can be used to
create a DNA fingerprint (DNA profile)
can identify a person because the DNA of an individual is
unique (except for identical twins)
Restriction Fragment Length Polymorphism (RFLP)
1. chromosomal DNA is treated with restriction endonucleases
2. analyzed by gel electrophoresis
The unique band pattern on the gel can be used as a method
of identification if compared to
band patterns from an individual of
known identity.
Short Tandem repeat profiling (STR)
STRs are repeating short sequences of DNA in the genome that
vary in length between individuals
The length depends on how many copies of a particular STR
are present
Using primers and PCR, the STRs of an individual are amplified
and then separated by gel electrophoresis. Each STR fragment
is fluorescently labelled, and the fluorescence emitted can be
analyzed by a computer.
Short Tandem repeat profiling (STR)
Using primers and PCR, the STRs of an individual are amplified and
then separated by gel electrophoresis.
Each STR fragment is fluorescently labelled, and the fluorescence
emitted can be analyzed by a computer.
Produces a series of peaks that
represent STRs of differing molecular
mass and, therefore, differing lengths.
Each individual has a unique series of
peaks in their STR profile.
F. DNA Sequencing
a method for determining, base by base,
the nucleotide sequence of a fragment of
DNA
developed by Frederick Sanger in 1977
done manually
Sanger Sequencing (dideoxy sequencing)
DNA replication
DNA replication requires:
DNA template
primer
DNA polymerase
Deoxynucleotides
(A,T,C, and G)
3’ OH group is required for DNA
elongation
Sanger Sequencing
Dideoxynucleotides lack a –OH group at the 2’ and 3’ carbons on the ribose sugar
THUS, DNA synthesis terminates when one of four possible dideoxynucleotides are incorporated
Produces DNA fragments of different lengths
DNA replication requires:
DNA template
Short primer
DNA polymerase
Deoxynucleotides
(A, T, C, and G)
3’ OH group is required for
DNA elongation
Sanger Sequencing requires:
4 reaction tubes, each contains:
DNA template
Short primer (radioactive labelled)
DNA polymerase
Deoxynucleotides
(A, T, C, and G)
One of four dideoxynucleotides in
low concentration
(A, T, C, or G)
Different lengths of DNA will be
produced
Steps in Dideoxy Sequencing
1. The DNA to be sequenced is denatured, and a primer anneals to
the 3′ end of the region to be sequenced.
2. Four separate reaction tubes are prepared. Each contains:
• denatured DNA with primer
• deoxynucleotides (A, T, C, and G)
• DNA polymerase
• plus one of the four dideoxynucleotides (ddG, ddC, ddA, or ddT)
Steps in Dideoxy Sequencing
3. DNA synthesis proceeds in each
reaction tube. DNA fragments of
different lengths are produced after
the incorporation of
dideoxynucleotides.
Steps in Dideoxy Sequencing
4. Each reaction tube’s fragments are
separated using gel electrophoresis.
5. The gel is read from bottom to top
to determine the sequence
6. The sequence read from the gel is
the synthesized strand. The
sequence of the original template
DNA is complementary to this
sequence.
Automated DNA Sequencing
New and efficient methods were developed for the Human
Genome Project.
The old Sanger sequencing was limiting for scientists could only
read 300 nucleotides at one and it took time to create and
separate the four reaction tubes.
Automated DNA Sequencing
New Method:
More bases can be read
Dideoxynucleotides are labelled with their own color of dye tags
All dideoxynucleotides are added to one reaction tube only
In gel electrophoresis, only one lane is required for the only
reaction tube and a laser lights up the tags in the gel
Photodetectors are used to identify the colour and the fragments
can be analyzed.
Automated DNA Sequencing
Each peak can be
interpreted as a nucleotide
in the DNA sequence.
The sequence of the
original template DNA is
complementary to this
sequence.
G. Making Sequence-Specific Mutations
In 1976, Canadian scientist Michael Smith developed a
method called site-directed mutagenesis to study human-
made mutations and their cellular effects.
Site-directed mutagenesis is a method of specifically altering
the nucleotide sequence of a region of DNA.
This allows scientists to study the structure and function of genes
and proteins.