Introduction to DNA
Chapter 4
4.1 DNA Structure and Function
Manipulations of DNA and RNA are center for biotech research“Central Dogma” Small sections of
DNA (genes)
mRNA
proteins
Transcription
Translationsat ribosome
4.1 DNA Structure and Function
Purines
Pyrimidines
4.1 DNA Structure and Function
Made of 4 nucleotide monomers that contain 4 nitrogenous basesForm double helix (2 sides) of repeating nucleotides, several million pb in length ie…same DNA structureA from one strand always with T from another and C from one strand always with G from another and ….so equal amounts of eachA,T, C, G are stacked 0.34 nm apart with 10 nitrogen bases per complete turn of the helix
Number of DNA strands in the cells of an organism (i.e. # of chromosomes)
Length in bp of DNA strand
Number and types of genes
Shape of DNA strand (circular or linear chromosomes)
Similarities in DNA Molec. Among Organisms
Variations in DNA Molec.
4.2 Sources of DNA
Prokaryotic Bacterial cell culture
EukaryoticMammalian cell culture
Viral
4.2 Prokaryotic DNABacteria cells ex E.Coli
Do not contain nucleus or membrane bound organelleDNA is floating in cytoplasmTypical bacterium contain only one circular long DNA (a chromosome)Some bacteria have extra small rings of DNA (plasmids)
4.2 Prokaryotic DNA
Most familiar plasmids are R plasmidsContain antibiotic resistance genes
Bacteria with these genes can survive antibiotics that would normally kill them
Bacteria can transfer plasmids ie genetic info between themselves
So, genes for antibiotic resistance can be transferred btw bacteria and lead to deadly antibiotic resistant disease causing bacteriaGives bacteria a way of evolving
4.2 Prokaryotic DNA
Different bacteria have different plasmidSome bacteria have more than one kindSome bacteria have nonePlasmids are small and easy to extract from cells
Often used as rDNA vectors to transform cells
4.2 Prokaryotic DNA
DNA fragments (genes) can be cut and pasted into plasmid vector
Recombinant plasmid can then get introduced to cellCell will read DNA code on the r-plasmidThen start synthesizing proteins coded for the geneSame procedures Genentech, Inc used to produce human insulin from E-Coli
Gene Expression Process
Prokaryotic DNA’s gene expression (genes turned on or off ) is simple with only few controls
An operon (1 or more genes and their controlling elements)RNA polymerase (enzyme that synthesize mRNA molec.) attach to DNA segment at a promoter region of operon this “turns on” gene RNA polymerase works its way down DNA strand to structural gene to built mRNAmRNA is decoded into a peptide at a ribosomeOperator can then “turn off” the gene
Operon
Section that codes for mRNA which later get translated to proteins
Gene Expression Process
In case of regulatory molec. Attach at operonOperon get “turned off” b/c RNA polymerase is blocked from continuing down the strand to the geneNo protein is producedBlocking and unblocking is how bacteria make certain proteins certain timesExample: Lactose
Bacterial Cell Culture
To manipulate bacteria DNA, cells are neededSome bacteria prefer
Liquid medium (Broth)Solid medium (agar)Grow well in either
AgarMixture of water and protein molec.Preparation:
Powdered agar and water are mixed, heated until agar completely suspendedAgar is sterilized at high temps (121C or higher) and high pressure (15 psi or higher) cooled to 65C and poured under sterile condition in Petri dishes Agar cools and solidifies in 15-20 minPlates can be used after 24 hr.
Broth
Also water and protein molec.Cultures grow as suspension of million of floating cells
4.2 Eukaryotic DNAFrom protist, fungi, plants, and animal cellsSame as prokaryotic
Same nucleotides (A, C, G, T)Same double helix of repeating nucleotidesEach antiparallel strand bound to the other by H-bonds
Different from prokaryoticPacked into chromosomes (regulated and expressed different from bacteria)Several chromosomes/cell vs. only 1/cellEach chromosome is single, linear, very long molec of DNALack of operators
Chromosomes range 4-100 or moreHuman have 46 chromosomes, fruit flies 8, ferns more than 1000Amount of DNA/cell is not directly related to organism’s complexity
Gene Expression
Controlled differently than prokaryotes
Eukaryotic gene expressed at very low levelIncrease in expression happen when enhancer (section of DNA) molec. interact with RNA polymerase or with enhancer DNA regions
Molecules attach to DNA and increase gene transcription
Gene Expression
Gene contains promoter region where RNA molec. recognize the geneRNA polymerase attach and move down DNA molec. To get to structural genesAt structural genes RNA polymerase builds a complementary mRNA transcript from one side of DNA strandThe enzyme transcribes the entire gene until reaches a termination sequence
Gene Expression
ProkaryoticmRNA transcript is immediately translated into polypeptide at a ribosomeNo introns and exons at structural genesOperator to regulate gene expression
EukaryoticmRNA is often modified before translationStructural genes are made of intron and exon sections
Exons: DNA sections that contain the protein codeIntrons: spacer DNA
No operator so produce molec. called transcription factors to turn genes onAlso regulated by way chromosome coiled (around histones proteins)
Coiling makes genes buried and RNA polymerase can not get to themDNA has to uncoil
Mammalian Cell Culture
Growing them more challenging that bacteria
Normally grown in broth culture
4.2 Eukaryotic DNAViral DNA
VirusesDo not have cellular structureCollection of protein and nucleic acid molec. that become active once they are within a suitable cellVery small, measure from 25-250 nmBased on type of cell they attach classified as:
Bacterial, plant, or animals
Have thick protein coat surrounding nucleic acid core of either DNA or RNA
Animal virus
Viral DNA
Within a cell:Nucleic acid of a virus is releasedViral genes are read by host cell enzymeDecoded into viral mRNATranslated into viral proteinsNew virus molec. are assembled and released and may infect other cells
Viral DNA
Viral DNA and RNA molec are short, easy to manipulateCalled vectors
4.3 Isolating and Manipulating DNA
Genetic engineering (G.E.):All modifications of DNA code of an organismProcess:
ID of molec produced by living things that could be produced more economically or easily through G.E. (ex. insulin)Isolation of gene for production of molec (insulin gene)Manipulation of DNA instructionHarvesting of molecule or product
Recombinant DNA Technology
Methods used to create new DNA molec by piecing together different DNA molec
When cells accept rDNA and start expressing the new genes by making new proteins….they are considered genetically engineered
Many items on market with rDNA technology
Ex recombinant rennin , chymosin
Site-Specific Mutagenesis
Process of inducing changes in certain sections of a particular DNA codeAccomplished through use of chemicals, radiation, virusesSometimes “directed”(on purpose by scientist to make better changes)
Ex: subtilisin marketed by Genecor international (tide)An enzyme (protease) that degrades proteins Added to laundry detergent to stains like blood, gravy
Gene Therapy
Process of correcting faulty DNA codes that causes genetic diseases and disordersCommon way to use virus to carry a normal gene into cells containing defective ones (gene replacement)
Correct diseases like Parkinson's, diabetes, cystic fibrosis (CF), and some cancers
CF:One of every 3000 babies born with itCauses build up of thick mucus that clogs the respiratory and digestive systems
Gene Therapy
In 2002:Modified cold virus used to transfer normal copy of the gene cystic fibrosis transmembrane conductance regulator (CFTR)To cells lining the noseCFTR gene is defective in CF patientsRegulates flow of Cl- ions into epithelial cells lining respiratory and digestive systemsThis method called adeno-assosiated virus (AAV) delivery system b/c uses modified adenovirus (cold virus)Worked only in small # still in clinical trials
4.4 Gel Electrophoresis
Uses electricity to separate charged molec. on a gel slab
Separation based on size, shape and chargeEx: DNA and RNA fragments, and proteins
Gel:Powdered agarose (carb. derived from seaweed)Dissolve in boiling buffer soln. Most common agarose is polyacrylamide (PAGE)Gel solidify and placed in get box and covered with buffer soln.
Gel Electrophoresis
Gel Electrophoresis
Gel Electrophoresis
Agarose used when separatingDNA pieces no smaller than 500bp and no larger than 25,000bpMade at a specific concentrations ranging (0.6-3%) figure 4.32High concentration for smaller molec
Gel Electrophoresis
Gel stains:Nucleic acids are colorlessMust be stainedDNA stains:
Ethidium bromide (EtBr)…orange when mixed with DNA under UV lightMethylene blue…dark blue…not as sensitive as EtBr viewed with white light
Gel Electrophoresis
Note: Only negative charge would run on this one
Only one DNA type
most common Sizing standard
Plasmid restriction digestionDNA sample from bacterial
chromosome RNA
Smears (thousands of different size molec in small concentration)
No nucleic acids
DNA so large will not load
Ex: eukaryotic genome
Homework
Sec 4.3 Review question 1, 2, 3, 4Sec 4.2 Review questions 1, 2, 3, 4Sec 4.4 Review questions 2, 3, 4Think like Biotech
2, 3, 6, 7, 8
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