Amount of DNA in l Hindlll bands:23.1 kb 240 ng9.3 kb95 ng6.6 kb 70 ng2.3 kb25 ngA student was given two samples, A and B. She took 3 ml of sample A and added 7 ml of water and 2 ml of sample buffer to it. That entire sample of 12 ml was loaded into lane 1 on the gel below. She took 3 ml of sample B and added 7 ml of water and 2 ml of sample buffer, and loaded that entire sample into lane 2.

1. What is the approximate amount of DNA in the band in lane 1? ____________2. What are the size of the DNA fragments in lane 1? ____________3. What is the size of the DNA fragments comprising the DNA band in lane 2? ____________4. What is the concentration of DNA in sample B? ____________

Which band contains more DNA? Which band is larger?

If I told you that the starting concentration of the DNA sample in lane 3 was 50 ng/ul, how many ul of sample were loaded?23,130 bp, 240 ng9,416 bp, 95 ng6,557 bp, 70 ng

4,361bp na

2,322 bp, 25 ng 2,027 bp, 20 ng

Lecture 3

Cloning DNA

Making a genomic library

Isolation of DNA from Vibrio fischeri

Lecture/Lab 3 Learning Objectives:By the end of lab today, you will be able to

Be able to briefly explain to a non-expert the importance of the development of recombinant DNA technology.

Explain the function of each reagent used during genomic DNA isolation and what bacterial component it acts on.

Predict (generally) what would happen if a particular reagent or step was skipped during DNA isolation.

DNA CloningIf you introduce a foreign piece of DNA into a another organism, and that piece of DNA gets copied when the host cells replicate, you will have a large number of cells all with identical copies of that piece of foreign DNA so you have cloned that piece of DNA, and can use the host cells to replicate it.

If that piece of DNA codes for a protein, it is also possible for the host cells to produce the protein.

What do you need to clone DNA?Source of DNA to be clonedgenomic DNA or cDNA (DNA copied from RNA)Vector to get foreign DNA into host organismPlasmid, virus, phage, etcType of vector you use depends on hostHost organismCan be bacteria, yeast, mammalian cell, etcBacteria easiest to use but there are sometimes issues expressing eukaryotic proteins in bacteria

Figure 1.1The basic steps in gene cloning.Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.

Why clone DNA?Study function of proteins etc. in cells.

Produce human proteins to use as drugs insulin, growth hormone

Make genetically modified plants that are resistant to pests or produce vitamins, etc

Gene therapy transfer good copy of a gene into a patient with abnormal copy

A chainB chainInsulin is a small protein consisting of an A chain of 21 amino acids linked by two disulfide (SS) bridges to a B chain of 30 amino acids. It is produced by beta cells in the pancreas, and is necessary for glucose uptake into certain tissues like liver, muscle and fat.

Type 1 diabetics, are absolutely dependent on insulin since their beta cells no longer secrete insulin, and even Type 2 diabetics may become dependent on insulin.Example: Cloning of human insulin

Prior to the production of insulin by recombinant DNA techniques, diabetics were injected with insulin isolated from cows and pigs.

In the 1970s,several groups attempted to identify and then clone the human insulin gene. Genetech however, won the patent, licensed it to Eli Lilly, who began producing human insulin in E. coli = Humulin

Recombinant DNA technology has also made it possible to manufacture slightly-modified forms of human insulin that work faster (Humalog and NovoLog) or slower (Lantus) than regular human insulin.

Other recombinant proteins

Human growth hormoneDwarfismFactor VlllBlood clottingHepatitis B vaccineAnti-TNF alpha receptor antibodyAutoimmune diseasesGolden riceVitamin A deficiencyChymosinCheese manufacturing

Creating a genomic libraryA Genomic library is a collection of clones representing the entire genome of an organism

1. Isolate genomic DNA from source2. Break up large pieces of chromosomal DNA into smaller pieces vectors have limits in terms of sizes of insert3. Cut open vector, and then ligate pieces of genomic DNA into vector4. Transform bacteria or other host cells with recombinant vectors to get individual clones = host cells that contain a single vector with the same piece of DNA5.Different clones will contain different pieces of DNA if different pieces of DNA in all clones assembled together, the whole genome of the organism can be recapitulated

Figure 1.3Cloning allows individual fragments of DNA to be purified.Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.

1. Isolate genomic DNA from bioluminescent bacteria Vibrio fischeri2. Digest genomic DNA with restriction enzyme SalI to create smaller DNA fragments3. Ligate genomic DNA fragments into plasmid vector pGEM that has also been digested and cut open with SalI4. Transform E. coli strain DH5alpha with recombinant plasmids and then culture on agar-ampicillin plates 5. Select individual clones to sequence inserts in plasmidsand6. Screen clones to see if any are producing proteins involved in bioluminescence

Creating a genomic library from Vibrio fischeri DNA

Isolation of genomic DNAGrow Vibrio fischeri cells in high-salt liquid media at room temperature overnight then centrifuge to pellet cells

Lyse cells openV. fischeri are gram-negative bacteria require several steps to break open because of cell envelope

Inhibit DNases use EDTA, pH 8.0

Get rid of all other components Proteins/lipids/sugars phenol/chloroform extractionRNA use RNase

Concentrate DNA using ethanol

Bacterial chromosome

Gram negative bacteria cell envelope

NucleasesNucleases are enzymes that either cut or shorten DNA or RNA strands by breaking the phosphodiester bondsExonucleases remove nucleotides from the ends of DNA moleculesEndonucleases cut DNA in the middle of the chain-Some endonucleases like DNase l are non-specific will cut DNA anywhere in sequence-Others, like Type ll restriction enzymes, will only cut DNA at specific sequencesBoth types of DNA nucleases require Mg2+ as a cofactor

Inhibit DNasesV. fischeri has lots of periplasmic nuclease activity so it is important to get rid of these proteins before they have a chance to digest our DNA! As you break open the bacteria, the DNases that are normally localized in the periplasmic space are in contact with chromosomal DNA and could digest it

So start DNA purification procedure by re-suspending cells in TES buffer:T = 10 mM Tris buffer, pH 8.0 - DNA more stable at slightly alkaline pHE = 1 mM EDTA to remove Mg2+ and inhibit DNases.S = sodium chloride need sodium for ethanol precipitation later in procedure

Lysing gram negative bacteriaAdd a chelating agent of divalent metals (e.g. EDTA) to disrupt outer membrane lipopolysaccharides. Mg2+ is bound to LPS (lipopolysaccharide layer) and stabilizes it. EDTA is a chelator that removes Mg2+ and destabilizes membrane.

2. Add lysozyme to break up peptidoglycan layer, which is critical for maintaining shape and rigidity of bacteria.(Peptidoglycan layer made of polysaccharides and amino acids lysozyme cleaves sugar component.)

3. Detergent SDS added to help lyse the cells by disrupting lipid bilayer also denatures some proteins

Removing other cellular componentsAdd RNases enzymes that digest RNA (lab 4)

Add proteases enzymes that digest proteins (lab 3 & 4) Proteinase K is actually more active in the presence of SDS, which denatures most proteins, and is more active at higher temperatures (up to 65C).

Phenol-chloroform extraction: both are organic solvents that will dissolve lipids and dissolve and denature proteins. Phenol equilibrated to pH 8 will cause lipids, protein, and polysaccharides to partition into organic phase also dissociates proteins from nucleic acids.Chloroform helps the two phases separate better and is easier to evaporate than phenol.

Figure 3.6Removal of protein contaminants by phenol extraction.Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.http://bitesizebio.com/articles/the-basics-how-phenol-extraction-works/

Concentrate DNAUsually use alcohol to precipitate nucleic acids out of solutionNucleic acids have limited solubility in alcohol solvents. ETOH is volatile and the pellets can easily be dried. will only work in presence of monovalent cationmonovalent cations shield negative charges and reduce repulsion DNA precipitates. http://bitesizebio.com/articles/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/

What reagents will we deploy to disrupt the cell wall and the membranes of these bacteria?

Discuss with your group____________________________________________

Explain to your nearest neighborHow will you purify DNA from proteins and lipids. Indicate the roles of the reagent(s) youll use.

How will you concentrate your DNA. Indicate the roles of the reagent(s) youll use.

~150ng/X = 50ng/uLX = ~3uL**Why clone? Study genome, produce protein for medicine, etc.*Mention source could be anything and that cDNA is copy of RNAMention that host could be plant, mammal, etc****Vibrio has genome of about 4.2 Mb explain Mb*EDTA, prot K, lysozyme, SDS*