Mary K.

Campbell
Shawn O. Farrell
international.cengage.com/

Chapter Thirteen
Nucleic Acid Biotechnology Techniques

Paul D. Adams • University of Arkansas

1
Purification and Detection of Nucleic Acids
• Gel electrophoresis is
a common technique
used to separate
nucleic acids.
• Based on motion of
charged particles in
an electric field

2
Purification and Detection (Cont’d)
• Radioactive labeling of sample used to detect products

• Label or tag allows visualization

• DNA undergo reaction that incorporate radioactive isotope into the DNA

• Autoradiography used to visualize image that has been exposed to

oligonucleotides that have been radiolabeled
• Fluorescence also used. Ethidium Bromide…can slip between DNA
bases, and it has different fluorescence characteristics as opposed to
when it is free in solution
• EtBr is used as stain for DNA on gels. EtBr is dangerous ( a
carcinogen)…new fluorescent dyes have been developed (SyBr Green
and Gold)

3
Restriction Endonucleases
• Nucleases- catalyze the hydrolysis of the
phosphodiester backbone of nucleic acids
- Endonuclease: cleavage in the middle of the chain
- Exonuclease: cleavage from the ends of the
molecule

• Restriction Endonucleases- Have a crucial role in

development of recombinant DNA technology

• Bacteriophages, viruses that infect bacteria, were

being studied when restriction enzymes were
discovered
4
Methylation of DNA

5
Restriction Endonucleases (Cont’d)
• Restriction endonuclease (RE) hydrolyzes only a specific
bond of a specific sequence in DNA

• Sequences recognized by RE read the same from left to right

as from right to left, known as palindrome

• Two As and 2 Ts between breaks in DNA strand which leave

sticky ends

• Sticky ends are joined by by hydrogen bonding between

complementary bases.

• Ligases reseal ends

6
Restriction Endonucleases and Their
Cleavage Sites

7
Action of DNA Ligases

8
Cloning
• Recombinant DNA- DNA molecules that contain covalently
linked segments derived from 2 or more DNA sources

• Sticky Ends can be used to construct Recombinant DNA

• DNA Ligase- seals nicks in the covalent structure

• Plasmid- small circular DNA that is not part of the main

circular DNA chromosome of the bacterium.

• Cloning- The process of making identical copies of DNA

9
Production of Recombinant DNA

10
Plamids
• How do we know which bacteria takes up the
desired plasmid?

• Selection- Each plasmid chosen for cloning has a

selectable marker that indicates that the growing
bacteria colonies contain the plasmid of interest

11
Plasmid pBR322
• One of the first plasmids used for cloning

12
Plasmids (Cont’d)
• As the technology to design plasmids improved,
regions were created that had many different
restriction sites in a small place

• This region is known as a multiple cloning site

(MCS), or polylinker

13
Cloning with pUC Plasmids

14
Blue/White Screening
• Basis for selection

• pUC plasmids contain lacZ gene

• lacZ gene codes for the α-subunit of β-

galactosidase, which cleaves disaccharides

• This procedure helps with selection

15
Clone Selection with Blue/White Screening

IPTG – isopropylthiogalactosie – lactose

analogue that also binds to lac repressor

X-gal - reagent that produces blue colour

when converted by β-galactosidase

16
Cloning Summary
• Cloning refers to creating identical populations
• DNA can be combined by using restriction enzymes
• The target DNA sequence is carried in some type of
vector
• The target DNA sequence is inserted into host
organism
• Organisms that carry the target DNA are identified
through a process called selection

17
Genetic Engineering
• When an organism is intentionally altered at the
molecular level to exhibit different traits, it has been
genetically engineered
• One focus of genetic engineering has been gene
therapy, where cells of specific tissues in a living
person are altered in a way that alleviates the affects
of a disease
• DNA recombination can occur in nature
• The reproductive power of bacteria can be used to
express large quantities of a mammalian protein of
interest, however, process can be complicated

18
Cloning Vectors
• Plasmid vectors pBR322 and pUC are cloning
vectors
• Vectors are used to insert foreign DNA and amplify it
• If we want to produce produce protein from the
foreign DNA, vectors are not good

19
Expression Vectors
• Have many attributes as cloning vector:
• The origin of replication
• A multiple cloning site
• At least one selectable marker
• Must be able to be transcribed by the
genetic machinery of the bacteria where it is
transformed
• Must have a transcription termination sequence

20
DNA Libraries
• Can we take all the
DNA of an organism
and clone it in chunks
of reasonable size
• The result of this is a
DNA library
• Several steps involved
in construction of the
library

21
Finding an Individual Clone in a DNA
Library
• After the library has been
constructed, the next
challenge is to find a
single desired clone out
of hundreds of
thousands, or millions
• Technique used to
select depends on
separating and annealing
complementary strands
• Known as Genomic
Library Screening
22
Finding an Individual Clone in a DNA
Library (Cont’d)
• RNA libraries not
constructed in the same
way
• RNA of interest is used as
template for the synthesis
of complementary DNA
(cDNA)
• Reaction catalyzed by
reverse transcriptase
• cDNA is incorporated into
vector, then process is
identical to the production
of genomic DNA library

23
Summary
• A DNA library is a collection of clones of an entire
genome
• The genome is digested with restriction enzymes
and the pieces are cloned into vectors, and
transformed into cell lines
• Specific radioactive probes to a sequence of interest
are reacted to filters that have copies of the bacterial
colonies in the library
• A cDNA library is constructed by using reverse
transcriptase to make DNA from the mRNA in a cell.
This cDNA is then used to construct a library similar
to a genomic DNA library
24
The Polymerase Chain Reaction
• It is possible to increase the amount of a given DNA
many times over without cloning the DNA

• This method of amplification is known as the

Polymerase Chain Reaction (PCR)

• Any chosen DNA can be amplified, and it does not

need to be separated from the rest of the DNA in a
sample before the procedure is applied

25
The Polymerase Chain Reaction (Cont’d)

26
DNA Fingerprinting
• DNA samples can be studied and compared by
DNA fingerprinting
• DNA is digested with restriction enzymes and
then run on an agarose gel
• When soaked in ethidium bromide, the DNA
fragments can be seen directly under UV light
• If greater sensitivity needed or if number of
fragments would be too great to distinguish the
bands, technique can be modified to show only
selected DNA sequences
• This begins with Southern blotting

27
The Southern Blot

28
Restriction-Fragment Length
Polymorphisms
• In organisms with two sets of chromosomes, a given gene on
one chromosome may differ slightly from the corresponding
gene on the paired chromosome

• These are known as alleles

• Organisms are homozygous when they have the same paired
chromosomes
• Organisms are heterozygous when they have different paired
chromosomes

• Restriction fragments of different sizes are obtained by

treatment with endonuclease. They are Restriction-
Fragment Length Polymorphisms (RFLPs)

29
The Basis for Restriction-Fragment Length
Polymorphism

30
Summary
• A DNA fingerprint is created by digesting DNA with
restriction enzymes, separating the pieces on a gel,
and visualizing some of the pieces by using labeled
probes

• Differences in DNA patterns between different

individuals are based on different base sequences of
their DNA

31
DNA Sequencing
• The nature and order of monomer units determine
the properties of the whole molecule
• The method devised by Sanger and Coulson for
determining the base sequences of nucleic acids
depends on selective interruption of oligonucleotide
synthesis
• A single-stranded DNA fragment whose sequence is
to be determined is used as a template
• The synthesis is interrupted at every possible site in
the population of molecules depending on the
presence of ddNTPs

32
DNA Sequencing (Cont’d)
• The incorporation of the ddNTP into the growing chain causes
termination at the point of incorporation

• The DNA to be sequenced is mixed with a short

oligonucleotide that serves as a primer for synthesis of the
complementary strand

• Gel electrophoresis is performed on each reaction mixture,

and a band corresponding to each position of the chain
termination appears

• The sequence of the newly formed strand, complementary to

the template DNA, can then be read from the sequencing gel
33
The Sanger-Coulon Method for Sequencing
DNA

34
Summary
• DNA can be sequenced by using several techniques,
the most common being the chain termination
method
• Dideoxy nucleotides are used to terminate DNA
synthesis. Multiple reactions are run with different
dideoxy nucleotide in each reaction mix
• The reactions produce a series of DNA fragments of
different length that can be run on a gel and the
sequence determined by tracking the different length
fragments in the lanes with the four different dideoxy
nucleotides

35