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.