Chapter 13Nucleic Acid Biotechnology Techniques

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Paul D. Adams • University of Arkansas

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

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

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

Methylation of DNA

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

Restriction Endonucleases and Their Cleavage Sites

Action of DNA Ligases

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

Production of Recombinant DNA

The Cloning of a Virus

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

Plasmid pBR322

• One of the first plasmids used for cloning

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

Blue/White Screening

• Basis for selection

• pUC plasmids contain lacZ gene

• lacZ gene codes for the -subunit of -galactosidase, which cleaves disaccharides

• This procedure helps with selection

Clone Selection with Blue/White Screening

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

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

Genetic Engineering (Cont’d)

• Human proteins can be made by bacteria, but process is not straight forward. e.g. human insulin

• An intron is a DNA sequence that codes for RNA that is eventually deleted in the processing of the mRNA that directs the synthesis of the protein

• Only the RNA transcribed from exons appear in the mature RNA

Protein Expression 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

• Instead, expression vectors are used

What is an Expression Vector?

• 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

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

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

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

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

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

The Polymerase Chain Reaction (Cont’d)

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

The Southern Blot

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)

The Basis for Restriction-Fragment Length Polymorphism

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

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

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

The Sanger-Coulon Method for Sequencing DNA

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

Genomics and Proteomics

• Knowing the full DNA sequence of the human genome allows for the investigation for the causes of disease in a way that has not been possible until now

• The proteome is a protein version of a genome

• Proteomics is the study of interactions among all the proteins in a cell

Microarrays

Summary

• As more DNA sequences become available, it becomes possible to compare these sequences

• Important medical applications are emerging, and new methods are making it possible to analyze large quantities of data

• The proteome is the protein version of the genome. It refers to all of the proteins being expressed in a cell

Summary

• DNA or protein microchips is a powerful technique being used presently, as thousands of samples of DNA or proteins can be applied and then checked for binding of biological samples

• The binding can be visualized by using fluorescently labeled molecules and scanning the chip with a computer (Figure 13.30). The pattern of fluorescent labels then indicates which mRNA or proteins are being expressed in the samples