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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