ANS/PLSS 433: Recombinant DNA Techniques--Cloning, Sequencing, PCR, etc.

I.	DNA and Gene Expression--The Basis for Molecular Biotechnology

	1.	DNA (Deoxyribonucleic acid)
		
		A.	All living cells contain the genetic material--DNA

			a.	Double Helix--Two strands
			b.	5'-3' direction
				i.	Sense strand
				ii.	"Top" strand
			c.	3'-5' direction
				i.	Antisense strand
				ii.	"Bottom" strand

		B.	Nucleotides make up the genetic code

			a.	Purines
				i.	Adenine--A
				ii.	Guanine--G
			b.	Pyrimidines
				i.	Cytosine--C
				ii.	Thymidine--T
			c.	A hybridizes (combines) to T 
					with two hydrogen bonds
			d.	C hybridizes to G with three hydrogen bonds
			e.	RNA also uses A,G,C, but substitutes 
				Uracil (U) for T
		
	2.	Gene Expression.

		A. 	Transcription 

			a.	DNA to RNA mediated by RNA polymerase 
			b.	Starts process at a promoter and stops at 
				a terminator.
			
		B.	Splicing

			a.	Eukaryotic genes contain exons (coding 
				regions) and introns (noncoding regions) 
				which are spliced out between transcription d translation.

		C. 	Translation 

			a.	Messenger RNA (mRNA) to protein is 
			b.	Mediated by ribosomes.  
			c.	mRNA must contain a ribosome binding 
				site just upstream from the initiation 
				codon (AUG).  
			d.	Codons are nucleotide triplets that code 
				for specific amino acids
			e.	Anticodons on tRNA recognize codon
			f.	Amino acids connected to tRNA polymerize 
				(combine) to form peptides
			g.	The ribosome continues to translate the 
				mRNA to protein until a stop codon 
				(UAA, UAG, or UGA) is encountered.

	3.	Post-translational modifications.  
	
		A. 	Many proteins contain a targeting or signal 
			sequence which directs their transport across 
			membranes.  
			
		B. 	Many proteins are formed as proproteins, have 
			disulfide bonds which stabilize the protein and 
			form secondary structure, and/or are glycosylated 
			for proper function. 

II. 	Genetic Engineering 

	1.	Genetic Engineering:  The technique of removing, 
		modifying, or adding genes to a DNA molecule.

	2.	Tools
		
		A.	Restriction Enzymes (RE, also called restriction 
			endonucleases)
			a.	"Genetic scissors" to cut DNA at specific 
				places--restriction sites.  
			b.	Restrictions sites are usually 
				palindromic DNA sequences
			c.	REs produce complementary sticky ends 
				or blunt ends.  
			d.	Sticky ends are preferred.
		B.	DNA Ligase
			a.	Molecular "Glue"
			b.	Adheres cut DNA back together  


	4.	Applications of Genetic Engineering:

		A. 	Genetic components (whole genes or parts of 
			genes) can be reconstructed into unique 
			combinations not easily achieved by natural 
			selection, or synthetic genes can be 
			constructed--Recombinant DNA.

		B. 	Recombinant DNA can be introduced or 
			reintroduced into bacteria, plants, or 
			animals.

			a. 	Bacteria to Plant:  
					Bt toxin, Herbicide resistance
			b. 	Plant to Bacteria:  
					Seed storage proteins, Enzymes
			c. 	Plant to Plant:  
					Chitinases, Promoters, 
					Herbicide Resistance.
			d. 	Bacteria to Bacteria: 
					Bt toxin, Herbicide resistance, etc.
			e. 	Animal to Plant:  
					Insulin, Antibodies, Pharmacologicals
			f. 	Animal to Bacteria:	
					Hormones, Immunity factors, etc.
			g. 	Animal to Animal:  
					Hormones, Pharmalogicals, 
					Gene Therapies.

	4. 	If an organism integrates recombinant DNA into their genome 
		or genetic make-up, they are transgenic.


III.	Cloning in Bacteria

	1. 	To propagate a DNA fragment, the gene must be supplied 
		with an origin of replication (ori), which acts as 
		a recognition site for DNA polymerase 1.  
		
	2.	Using recombinant DNA technology, we can insert the DNA 
		of interest in a cloning vector, which is usually in the 
		form of a plasmid. 
 
	3.	Plasmids have 3 common features:
		A.  	Oris capable of replicating in bacteria and yeast.			
		B.  	Selectable markers such as antibiotic resistance genes.
		C.  	Unique recognition sites for restriction enzymes.
		
			--Many cloning vectors also contain specific RNA polymerase 
				promotors so they can also be used as transcription 
				vectors. 

	4. 	To insert a DNA fragment into a plasmid, the DNA is 
		excised from its genome with one or two specific REs.  
		
	5.	The plasmid is also cut with these specific REs.  
		The DNAs are mixed and ligated, using DNA ligase, 
		to create recombinants or chimeras.

	6.	The chimeras are then cloned into a bacteria, usually 
		E.coli, by transformation (natural DNA uptake).  
		
	7.	The bacteria are then grown and the cloned DNA is 
		propagated.  

III.	Expression of Cloned Genes

	1. 	Shotgun cloning.  

		A.	The entire genome of one organism is 
			digested with REs and ligated into plasmids.  
		B.	Each plasmid gives rise to a transformed bacterium, 
			which produces an individual colony on an 
			agar plate, which contains one cloned bacteria
			a.	1000 clones from a bacterium, 
			b.	10 million from a plant or animal cell.  
		C.	However, even if you could identify the right cells,
			they probably still wouldn't express the gene.
			a.	Plant and animal genes are rarely expressed
				in bacteria because splicing doesn't occur 
				in prokaryotes
			b.	Cloning into yeast is one alternative
				--Very Inefficient

	2. 	cDNA cloning. 
		
		A. 	Copy DNA (cDNA) from spliced mRNA is synthesized 
			using Reverse Transcriptase (RT).  
		B.	The resulting cDNA can then be cloned and 
			expressed in prokaryotic cells

	3. 	Synthetic Genes cloning  
		
		A.	When the amino acid or nucleic acid sequence of 
			a gene is know can be resynthesized from 
			deoxyribonucleotides to form oligonucleotides.  
		
		B.	Combinations of oligonucleotides can be joined 
			to form whole genes and cloned into bacteria.

	4.	Consideration of Post-translational Modifications

		A.	Sequences need to be added if you want this 
			protein to be exported from a cell 
			(especially for expression in eukaryotic cells).  

			a.	They may need to be removed to get 
				heterologous expression
			b.	Protein must be extracted from 
				the cells.

		B.	Proproteins expressed in prokaryotes must undergo 
			an in vitro assembly

		C.	In vitro glycosylation is needed when eukaryotic 
			proteins must be glycosylated. 

IV.	Selection of Recombinants
	
	1. 	Complementation of a nutritional defect 
		(Protein Activity Screening).

		A.	Plasmid expresses nutritional enzyme that 
			recipient is missing.
		B.	lacZ operon which metabolizes galactose 
			is most common Select White colonies from Blue

	2. 	Resistance to antibiotic or herbicide.

		A.	Plasmid expresses antibody or herbicide resistance

	3. 	Immunochemical screening.
		
		A.	Uses antibody to find "clones" expressing protein.

	4. 	Nucleic acid hybridization.
		
		A.	Uses nucleic acid probes to find "clones" 
			having genes.

	5. 	Verification of insert.

		A. 	Restriction site mapping
			a.	Cut at Unique sites

		B. 	DNA Sequencing
			a.	Sanger, Dideoxy-mediated chain termination 
				(Most common)
			b.	Maxam and Gilbert, Chemical Degradation 
				of DNA Method
			c.	PCR Methods becoming more common

V.	Polymerase Chain Reaction

	PCR is revolutionary technique in genetic engineering which enables 
		specific DNA sequences to be amplified a thousand- to a 
		million-fold.

	1. 	Two oligonucleotide primers are required to hybridize or 
		anneal to opposite strands of the target sequence.  
		The sequence of interest will lie between the two primers.
		a. 5'-> 3' primer--to "bottom" strand.
		b. 3'-> 5' primer--to "top" strand.

	2. 	Taq DNA polymerase will then synthesize DNA between the 
		products in a process called primer extension doubling 
		the DNA segment.

	3. 	The strands will then be denatured by heat, and the primers will 
		be again annealed to all the single stranded DNA.  
		Taq is used because it does not get inactivated during the 
		denaturation steps.

	4. 	The process is repeated for multiple cycles to get exponential 
		accumulation of the specific DNA fragment
		--10 cycles will amplify 1000-fold.

	Applications of PCR in cloning:

	1. 	Genes can be amplified before being put into cloning vector to 
		eliminate need for sensitive screening methods.

	2. 	Can combine with reverse transcriptase to synthesize and 
		amplify cDNA from mRNA.

	3. 	Can recombine or splice DNA molecules at precise junctions 
		irrespective of nucleotide sequences ant the recombination 
		site and without the use of REs or ligase
			--Splicing by Overlap Extension (SOE).

VI.	Maximizing Gene Expression

	1. 	Copy number per cell affects expression.
	2. 	Promotor strength determines mRNA abundance.
	3. 	RBS and Terminator determine translation efficiency.
	4. 	Codon choice determines translation efficiency.
	5. 	mRNA stability or half-life determines mRNA abundance.
	6. 	Proteolysis determines amount of protein product accumulation.


Conclusion:  

	The techniques of molecular genetics (genetic engineering) 
	can enable expression of foreign genes across wide evolutionary 
	distances.  Care must be taken to modify appropriately to maximize 
	expression.