1. Nucleic Acid Biotechnology Techniques
Chapter 13

2. 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

3. 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
Isotope – p32 and S35. After exposure the gel is placed in contact with X-ray film.

4. Detection of Nucleic Acids
Fluorescence
Ethidium bromide intercalates between bases
Under UV light glows orange

5. 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
Nucleases are specific to DNA and some can be specific to RNA. One group of nucleases is RE.

6. Methylation of DNA

7. 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

8. Resealing by DNA ligase
Sticky ends are joined by hydrogen bonding between complementary bases.
Ligases reseal ends

9. 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

11. 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

12. Transformation Bacteria take up recombinant DNA Heat shock method
Electroporation
Transformed bacteria – scaled up

14. 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
Origin of replication and replicated independently of the chromosomal DNA.

15. 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
pUC – universal cloning plasmid

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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 have a transcription termination sequence

22. 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

23. Producing Large Numbers of Transformed Cells

24. 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

25. 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

26. 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

27. 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
RNA libraries not constructed in the same way

28. 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

29. 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

30. 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

31. 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

32. 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

33. Fig

34. 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

35. 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.

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