Nucleic Acid Biotechnology Techniques

Chapter 13

Separation techniques of Nucleic Acids

• Gel electrophoresis -used to separate nucleic acids based on charge and size.

• Proteins – SDS PAGE

• Done in an electric field

Detection of Nucleic Acids

• Radioactive labeling of sample used to detect products

• Label or tag allows visualization

• DNA undergoes reaction that incorporates radioactive isotope into the DNA

• Autoradiography used to visualize image that has been exposed to radiolabeled oligonucleotides

Detection of Nucleic Acids

• Fluorescence

• Ethidium bromide intercalates between bases

• Under UV light glows orange

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- Led to discovery of restriction enzymes

Methylation of DNA

Restriction Endonucleases

• Restriction endonucleases (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

• Sticky and Blunt ends

Resealing by DNA ligase

• Sticky ends are joined by hydrogen bonding between complementary bases.

• Ligases reseal ends

Recombinant DNA Technology

• Recombinant/Chimeric 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

What is Cloning?

• 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

Transformation

• Bacteria take up recombinant DNA

• Heat shock method

• Electroporation

• Transformed bacteria – scaled up

pBR322

• One of the first plasmids used for cloning – E.coli

• Foreign DNA must be inserted at unique restriction sites

• Confers resistance to two antibiotics – Tetracycline and ampicillin

Plasmids

• 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

Selection

• 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 bacterial colonies contain the plasmid of interest

Clone Selection with 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

Cloning Summary

• Cloning refers to creating identical populations• DNA can be combined by using restriction

enzymes + Ligases• The target DNA sequence is carried in some

type of vector/plasmid• The target plasmid 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 - 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

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 protein from the foreign DNA - Expression vectors

What is an Expression Vector?• Have many attributes as

cloning vector:

- The origin of replication

- A multiple cloning site

- At least one selectable marker

What is an Expression Vector?

• Must be able to be transcribed by the genetic machinery of the

bacteria where it is transformed

• Must have a transcription initiation and termination sequence

• Ribosomal binding site-translation

Producing Large Numbers of Transformed Cells

DNA libraries

• All the DNA of an organism - clone it in chunks of reasonable size

• The result of this is a DNA library

• Several steps involved in construction of the library

How do we find the piece of DNA we want in a library?

• Genomic Library Screening

• A nitrocellulose disc is put on the dish and removed

• Disc treated with denaturing agent to unwind DNA

• DNA is permanently fixed to disc by treatment with heat or UV light

How do we find the piece of DNA we want in a library?

• Expose DNA on disc to a solution that contains single stranded complementary DNA or RNA (radioactive probing)

• Wash the disc• Identify the colonies

Making cDNA library

• RNA of interest is used as template for the synthesis of complementary DNA (cDNA)

• Reaction catalyzed by reverse transcriptase

• cDNA is incorporated into vector

• cDNA library construction is identical to 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

• 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

Polymerase Chain Reaction

• It is possible to increase the amount of a given DNA many times over without cloning the DNA

• Any chosen DNA can be amplified, and it does not need to be separated from the rest of the DNA in a sample

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 – can be seen directly under UV light

Southern blotting

• 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

Sequencing

• 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

Fig. 17.11

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

Open book take home quiz for 30 points

• 1. Name the two kinds of gels used in electrophoresis and what molecules do those separate. Explain the original charges of those molecules and in which direction do they move in an electric field. In other words explain the effect of charge and size on the biomolecules.

• 2. What is methylation of DNA in Bacteria?• 3. Write names of any two enzymes and the name of the bacteria from

which it has been extracted. Explain how these enzymes have been named or their naming procedures.

• 4. Draw a recombinant DNA plasmid showing the sites of BamHI and HindIII along with same RE sites on your DNA of interest.

• 5. Explain the whole procedure of PCR along with factors required to run a PCR reaction.

• 6. Explain the procedure of RNA interference. • 7. Explain the Agrobacterium transformation done in plants.

• This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis:

• against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and

• against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.