Chapter Thirteen Nucleic Acid Biotechnology Techniques Paul D. - - PowerPoint PPT Presentation

Mary K. Campbell Shawn O. Farrell

• Chapter Thirteen

Nucleic Acid Biotechnology Techniques

Paul D. Adams • University of Arkansas

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Purification and Detection of Nucleic Acids

• Gel electrophoresis is

a common technique used to separate 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
• ligonucleotides 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

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Methylation of DNA

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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

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Restriction Endonucleases and Their Cleavage Sites

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Action of DNA Ligases

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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. circular DNA chromosome of the bacterium.

• Cloning- The process of making identical copies of DNA

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Production of Recombinant DNA

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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

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Plasmid pBR322

• One of the first plasmids used for cloning

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Plasmids (Cont’d)

• As the technology to design plasmids improved,

regions were created that had many different restriction sites in a small place restriction sites in a small place

• This region is known as a multiple cloning site

(MCS), or polylinker

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Cloning with pUC Plasmids

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Blue/White Screening

• Basis for selection
• pUC plasmids contain lacZ gene
• pUC plasmids contain lacZ gene
• lacZ gene codes for the α-subunit of β-

galactosidase, which cleaves disaccharides

• This procedure helps with selection

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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

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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
• The target DNA sequence is carried in some type of

vector

• The target DNA sequence is inserted into host
• rganism
• Organisms that carry the target DNA are identified

through a process called selection through a process called selection

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Genetic Engineering

• When an organism is intentionally altered at the

molecular level to exhibit different traits, it has been genetically engineered 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

• f a disease
• DNA recombination can occur in nature

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

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Cloning Vectors

• Plasmid vectors pBR322 and pUC are cloning

vectors

• Vectors are used to insert foreign DNA and amplify it
• 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

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Expression Vectors

• Have many attributes as cloning vector:
• The origin of replication
• A multiple cloning site
• 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

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DNA Libraries

• Can we take all the

DNA of an organism and clone it in chunks and clone it in chunks

• f reasonable size
• The result of this is a

DNA library

• Several steps involved

in construction of the in construction of the library

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Finding an Individual Clone in a DNA Library

• After the library has been

constructed, the next challenge is to find a challenge is to find a single desired clone out

• f hundreds of

thousands, or millions

• Technique used to

select depends on separating and annealing complementary strands

• Known as Genomic

Library Screening

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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

• f complementary DNA

(cDNA)

• Reaction catalyzed by

reverse transcriptase

• cDNA is incorporated into

vector, then process is identical to the production

• f genomic DNA library

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Summary

• A DNA library is a collection of clones of an entire

genome

• The genome is digested with restriction enzymes
• 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 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

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

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The Polymerase Chain Reaction (Cont’d)

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DNA Fingerprinting

• DNA samples can be studied and compared by

DNA fingerprinting

• DNA is digested with restriction enzymes and
• 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 fragments would be too great to distinguish the bands, technique can be modified to show only selected DNA sequences

• This begins with Southern blotting

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The Southern Blot

28

Restriction-Fragment Length Polymorphisms

• In organisms with two sets of chromosomes, a given gene on
• ne 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)

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The Basis for Restriction-Fragment Length Polymorphism

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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 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 their DNA

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DNA Sequencing

• The nature and order of monomer units determine

the properties of the whole molecule

• The method devised by Sanger and Coulson for
• 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 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
• ligonucleotide 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 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

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The Sanger-Coulon Method for Sequencing DNA

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Summary

• DNA can be sequenced by using several techniques,

the most common being the chain termination method 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 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

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