Week 1 all

What is a nitrogen base

Nitrogen bases = attached to deoxyribose sugar. Are the 4 building blocks of life 

1. Adenine  

2. Cytosine  

3. Guanine 

4. Thymine 

Purines = bases with a double-ring structure 

• Ex: G & A 

Pyrimidines = bases with a single ring structure 

• Ex: C & T 

What is a nucleoside

Nucleosides = A nitrogen base bound to an unphosphorylated sugar  

• When the ribose sugar is phosphorylated... 

  • Mono = nucleoside 

    • Ex: Adenosine monophosphate (AMP) 

  • Triphosphate = nucleotide 

    • Ex: Adenosine triphosphate (ATP) 

  • di-phosphate = 2 phosphorylation 

What is a nucleotide?

Nucleotides = essential building blocks of DNA and RNA, composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and a phosphate group 

What is a nucleic acid? What is it’s structure

Nitrogen bases attached to a deoxyribose sugar form a polymer with the other deoxyribose sugars of other nucleotides via phosphodiester bonds 

Nucleic acid = a macromolecule made of nucleotides bound together by the phosphate and hydroxyl groups on their sugars 

• Grows by the attachment of 5’ phosphate group of an incoming nucleotide to the 3’ hydroxyl group of the last nucleotide on a growing chain 

  • Gives the chain polarity (5’ & 3’ end) 

• Hybridization = formation of hydrogen bonds between 2 complementary strands of DNA 

What are the steps of DNA replication

1. Unwind the DNA via helicase 

2. Primase adds the primer  

3. DNA elongation via DNA polymerase  

   • Makes leading and lagging strand 

4. DNA ligase seals nicks and joins strands 

What is polymerase

Polymerase = responsible for polymerizing the nucleotide chains 

• Uses a guide/template strand to know what nucleotides to add to a chain 

What is exonuclease

Exonuclease = degrade DNA from free 3’ hydroxyl or 5’ phosphate ends 

• Don't work on closed/circular DNA 

• protects the sequence of nucleotides  

What is endonuclease

Endonuclease = break the sugar-phosphate backbone of DNA 

What is ligase

Ligase = an enzyme that forms phosphodiester bonds between existing DNA strands 

• Catalyzes the formation of a phosphodiester bond between adjacent 3’ hydroxyl and 5’ phosphoryl nucleotide ends  

What is nuclease

Nuclease = natural components of cellular lysates 

• Important to eliminate or inactivate when preparing nucleic acid specimens for clinical analysis 

What is helicase

• analysis 

Helicase = unwinds and untangles DNA for replication 

• The release of DNA for transcription, replication, and recombination without tangling is brought about through cutting and re-closing of the DNA sugar-phosphate backbone 

What is methyltransferase

Methyltransferase = catalyze the addition of methyl groups to nitrogen bases, usually adenines and cytosines in DNA strands 

What is gel electrophoresis?

Electrophoresis = movement of molecules by an electric current through a matrix/gel 

• DNA is negatively charged (because of the phosphate backbone) so it moves towards the positive pole 

• DNA travels at speeds inversely related to its size 

  • Big molecules go slower (don’t migrate far in gel) 

  • Small molecules go faster (further in gel) 

What are the principles of electrophoresis

Principles:  

1. Determine method for separation 

   • Type of gel/matrix 

   • Concentration (%) 

   • Running parameters (time/voltage) 

2. Select molecular weight marker (ladder) 

3. Loading  

   • Prepare samples (loading dye) 

   • Load wells/column & document loading order 

4. Perform electrophoretic separation 

5. Visualize and document results 

   • Stain 

   • Chemiluminescence/UV/fluorescence 

What’s the difference between agarose and polyacrylamide gels

Agarose

Polyacrylamide

Very safe material/easy to work with  Can be melted/remelted  Ran in a horizontal format  Lower resolving power  Not as good separating small molecules  Good for separating larger fragments (very porous)  Made from seaweed & agar components  Concentration used: 0.5-5%  Concentration determines size of the spaces of the gel: High conc. = smaller pores (smaller DNA molecules)  Low conc. = bigger pores (larger DNA molecules)

Components can be toxic   Usually ran vertically  Finer size resolution (small DNA)   DNA sequencing, capillary electrophoresis (1 base pair difference)  Use for separating small fragments (& single stranded DNA)  Can resolve 1bp size diff.  Often used to sequencing/mutation analysis  Protein electrophoresis (western blotting)  Concentration used: 3.5-20%

• Gels are porous like a sponge (allowing DNA to squeeze through with the electric field/matrix sieve 

• The concentration of gel/buffer affects the resolution of fragments of different size ranges 

What’s the difference in agarose and polyacrylamide gel prep.

Agarose prep	Polyacrylamide prep
Measure agarose  Determine % agarose conc. needed  Dilute with buffer to proper concentration  1X buffer to 30mL  Heat solution to dissolve  Cool to 55-60°C  Pour gel into casting tray  Add dye to sample before  Dye can affect mobility of DNA  Allow to polymerize   Remove comb, pour buffer, & prepare to load  Run gel at room temp. (4-10V/cm)	Dissolve acrylamide powder to make a stock solution  Acrylamide = potent neurotoxin that’s slightly permeable to latex gloves  If gel is denatured, urea is added to suppress base pairing   Add bis-acrylamide as a cross-linker  Polymerization is initiated by the addition of ammonium persulfate  Polymerization is stabilized with TEMED  Run gel at high voltages (low temp.)

What are the buffers that can be used in electrophoresis

Buffers:  

• Carries the current and protects the samples during electrophoresis 

• Typically comes as 10X or 50X stock 

  • Dilute to 1X for working solution 

Tris acetate EDTA (TAE) = DNA moves faster, but buffering capacity is smaller  

Tris borate EDTA (TBE) = better buffering capacity, DNA moves slower 

What’s the difference between TAE and TBE

TAE	TBE
Alkaline pH  Easy to recover DNA  Used for large fragment resolution (over 10,000 bp)  BIG FRAGMENTS  May require re-circulation since can be easily exhausted  Most common buffer used for agarose gels	Alkaline pH  Avoid using if DNA is to be purified form the gel  Can't purify DNA from gel) Good for small fragment resolution (5-10,000bp)  Very high buffering capacity  Also used for acrylamide gels (esp. DNA sequencing)  Versatile

* Both buffers can be used interchangeably for PCR/many molecular diagnostic applications 

 

What is the purpose of a loading dye

Loading dye = Gives color to DNA for easier visualization 

• Makes DNA denser than water so it sinks to bottom of well (weighs down) 

• Has tracking dyes that separate during electrophoresis to indicate progress of electrophoresis 

• Allows for visualization while loading 

What’s the purpose of nucleic acid stain and detection reagents

make DNA visible 

What is the purpose of a molecular weight marker (ladder)

Molecular weight markers (ladders) = a concentrated control stock of DNA fragments of known size 

• Necessary for determining the actual size of the DNA bands in your finished gel 

• Included in every gel 

What are the general type of equipment used for electrophoresis

• UV light box (transilluminator) 

  • Gel documentation systems 

• Well combs = used to make wells when casting gel 

• Microwave = used to heat agarose 

• Casting tray = used to make gel 

• Gel box = runs reaction 

• Gel power supply = powers reaction 

What’s the difference between the 3 nucleic acid application detection systems (ie. gelred)

Ethidium bromide	SYBR Green	Gel red
Can be used with a 254nm transilluminator  Intercalates DNA  CARCINAGEN  When EtBr is bound to DNA, it’s fluorescence intensity is greatly increased  Has low affinity for ssDNA  When added to gel before run, DNA takes 15% longer to run  Can be added to the gel (slower) or used as a post-stain (extra step)	Good for DNA & RNA  Can be added to run or post-stained  Fluorescent stain that can be used on a transilluminator  Good on both agarose and polyacrylamid dsDNA  ssDNA & RNA	Safer alternative to EtBr  Non-cytotoxic and non-mutagenic   Stained gels can be discarded in regular trash  Uses same UV wavelengths as EtBr  More sensitive than EtBr  Less background staining, easier to see faint bands

How do you Calculate a sample mixture for loading onto an agarose gel (DNA, loading dye, water) 

From the total final desired volume, subtract the loading dye and water amount. From there, subtract the amount of DNA you will use 

• If you have the DNA conc. From the Nanodrop. And you want to use DNA that is Xng. You will divide the desired ng by the DNA conc. To get the volume of DNA to pipette in (ul).  

What is a problem with the shown image

No wells seen and no labeling 

What is a restriction endonuclease

Restriction endonucleases = recognize specific short DNA sequences 

• Originate in nature (by bacterial cells as a defense mechanism against foreign DNA –phage) 

  • Protect host by methylation of host DNA and cleavage of unmethylated DNA 

  • Named after the bacteria it comes from 

• 3 different types: Type I, II, III  

  • Most are type II = cleave at specific recognition sites. Only unmethylated DNA 

    • Recognize palindromes (in general) 

    • Can cut 1 of 3 ways (sticky or blunt ends) 

  • Type I: methylation/cleavage (3 subunits)   

    • cuts >1,000 bp away from binding site 

      • Ex: EcoAI 

  • Type III: methylation/cleavage (2 subunits)    

    • cuts 24-26 bp away from binding site 

      • Ex: HinfIII 

• Can cut 1 of three ways: 5’, 3’overhang, or blunt end 

• Measured in units (U) 

  • Check the compatibility of the enzyme with the buffer – not all buffers work with all enzymes (some enzymes can work with multiple buffers) 

  • Use 10U of RE per microgram (ug) of DNA 

• They're named after the bacteria they come from  

  • EcoRI = E. Coli (first one discovered) 

• Be aware of star acitvity 

 

What is the molecular diagnostic use of restriction endonucleases?

Molecular diagnostic use = can see if there is some sort of mutation because a change in bp won’t allow for the RE to bind anymore (maybe a different RE will bind) 

• If there is a mutation, the RE will not cut (sample will look like control) 

What' are the 3 types of ends an endonuclease can make

5’ overhang = sticky end because of anti-parallel nature 

3’ overhang = sticky end because of anti-parallel nature 

Blunt end endonuclease = leave no overhanging bases after separation because it is a palindromes

How do restriction enzymes cut DNA (#fragments)

Restriction enzymes  

1. Recognize specific sequence (usually 4-6 nucleotides) 

2. Cut the DNA by breaking the phosphodiester bond on both strands 

3. Cutting results in 2 or more fragments 

   • Smaller recognition sequences results in more fragments generated because it is easier to find a match for a short sequence 

4. Resolve fragments by gel electrophoresis 

* The number of times a specific sequence occurs in a given organism is approximated by... 

Genome size in nucleotides/4^n 

n = the length of the recognition sequence  

* Master mix should be on cold block and gently mixed 

What is restriction enzyme mapping?

Restriction Enzyme Mapping: After digesting the DNA with RE and resolving the fragments by gel electrophoresis... 

• Number of bands indicates the number of restriction sites 

• Size of the bands indicates the distance between restriction sites 

What are the detailed steps of restriction enzyme cutting process?

Detailed steps:  

1. Consult the enzyme data sheet for details  

   • It's important to find out the correct conditions for the enzyme that you’re using (usually provided by manufacturer) 

2. Set up master mix on cold block 

   • Mix gently by flicking, then briefly spin 

3. For human genomic DNA = RE reactions typically incubate at 37°C for 5-18hrs 

4. Enzymes often must be heat inactivated after reaction is completed 

   • Usually between 55-88°C for 20 mins. 

5. Analyze by gel electrophoresis 

What is star activity

Star activity = When RE cuts the DNA too many times, results in extra bands (RE GONE NUTS) 

• Heat inactivation required to stop RE from over cutting  

• On a gel, there will not be a DNA smear visible at the top of the gel because DNA is degraded 

What could have happened to this gel (top)

* If there is a DNA smear at the top of the gel, then the enzyme only possessed partial activity. You must check the reaction conditions because there may be inhibitors present. 

What are the parts of a blood specimen?

Plasma = may contain some genetic material (used for HIV)

Buffy coat = DNA (WBCs &platelets)

Eryhtrocytes = RBCs 

What are the steps of an organic DNA isolation? (w/purpose of each reagent)

1. Lyse the cell (using detergents/proteases) (break the cell contents open) 

2. Acidification if needed (via acetic acid) (if pH needs to be lowered) 

3. Mix lysate with PCI reagent 

   • Forming upper aqueous (DNA) & lower organic phase 

4. Separate aqueous phase  

5. Add ammonium acetate or sodium acetate to encourage precipitation (due to salts) 

6. Add 100% ethanol (promotes DNA precipitation because it’s insoluble in alcohol) 

7. Incubate at –20 to –70 (freezer) (further encourages DNA precipitation) 

8. Centrifuge & pour out supernatant 

9. Wash DNA pellet with 70% ethanol (dissolves salt and not DNA) 

10. Resuspend DNA in TE buffer or water (DNA dissolved and ready for use) 

What is the purpose and workflow of ethanol precipitation of DNA

A technique for purifying and concentrating DNA from an aqueous solution

• The 100% ethanol promotes DNA precipitation because it is insoluble in alcohol 

• The 70% ethanol dissolves the salts ONLY without dissolving the DNA  

* You first want to encourage the most DNA precipitation as possible, once this is achieved, the salt is removed so the DNA alone can be extracted 

1. Add Salt (sodium acetate) to neutralize DNA’s negative charge

2. Add cold 100% ethanol (precipitates DNA out of solution & cold enhances it)

3. Incubate at -20C (allow precipitation to complete)

4. centrifuge to form pellet of DNA

5. Wash with 70% Ethanol (dissolves salt and not DNA)

   • centrifuge again to repellet DNA

6. Air dry pellet to remove ethanol

7. resuspend DNA in TE buffer or nuclease-free water

What are the steps of solid phase DNA isolation? Purpose of each reagent

Qiagen 

1. Lysis using AL (L = lysis) buffer and proteinase K 

• Disrupting cells open & stops proteins that can degrade the DNA 

2. Incubation at 56 degrees 

• Accelerates protein breakdown (Proteinase K digests better) 

3. Addition of 100% ethanol 

• Encourages DNA precipitation 

4. Addition of AW1/AW2 (W = wash) buffers 

• First wash removes proteins/contaminants 

• 2^nd wash removes the salt/contaminants 

5. Elution with AE (E = elution) buffer 

• DNA released from silica membrane (DNA released for use) 

Compare/contrast the spin-column method to the magnetic bead (Chelex) DNA isolation method

Chelex reagent/resin = Used in DNA purification where Chelex beads bind to the cellular debris after cell lysis. Allowing the DNA to be in the supernatant (used in forensics) 

Spin-column = 

Magnetic (Chelex)	Both	Spin
Used in forensics for saliva & buccal cell specimens   Can also be used for fixed, paraffin-embedded specimens  DNA found in supernatant  Cells lysed via boiling  Beads bind to cellular debris  Chelex removes multivalent cations that can damage DNA (prevent enzymatic damage)  Automation friendly  High throughput	Rely on solid-phase extraction  High purity	DNA binds to solid column matrix under high salt conditions  Silica-based resin/columns  Nanogram yield of highly purified DNA  Salt/precipitation method with binding membrane  Cells lysed with protease  Low-medium throughput (manual)

What is Salting out

Salting out = inorganic DNA extraction = purification of nucleic acid by precipitating proteins and other contaminants with high salt at low pH 

• An alternative to using Phenol (toxic reagents) 

• Low-pH & high salt conc. Causes proteins to be precipitated and DNA left in solution. 

  • DNA is separated and then precipitated in isopropanol (ultimately resuspended in TE buffer/water) 

What are the steps to a DNA isolation using a Qiagen spin column

1. Lyse cells (detergent protease) 

2. Add to column, spin (DNA binds to matrix & waste flows through) 

3. Wash, spin (removes contaminants from column) 

4. Add elution buffer and spin (low salt will release the DNA from the column into a new clean tube) 

How are gel-based methods used to determine quality/quantity of DNA preparations?

• Quantity = intensity of gel bands 

  • Via densitometry 

• Quality = no smearing on gel & high molecular weight bands 

  • Excessive smearing means there is degraded DNA 

How are spectrophotometric methods used to determine quality/quantity of DNA preparations?

Spectrophotometric = instrument used to measure the absorbance of light at a particular wavelength (QUALITY + QUANTITY) 

• • Nucleic acids absorb light at 260nm  

  • Proteins absorb light at 280nm 

  • Expect a purified sample to have a high A260 and low A280 

• 260/280 ratio indicates QUALITY 

  • Low = protein contamination 

  • High = other contamination 

• Nanodrops give you the DNA concentration which gives you QUANTITY 

• Can't distinguish between DNA & RNA 

How are fluorometric methods used to determine quality/quantity of DNA preparations?

Fluorometric = Binding fluorescent dyes to DNA and detecting it via a fluorometer 

• More sensitive than spectrophotometric method (QUANTITY) 

  • Can distinguish between DNA/RNA/contaminants 

• Good for very SMALL amounts of DNA (smaller than nanodrop) 

• Not affected by phenol, EDTA, protein, and high salt contamination 

How do you calculate concentration and yield of DNA from a preparation

Concentration = amount/volume 

Yield = (starting DNA/RNA concentration) / (ending DNA/RNA concentration)

How do you read a spectrophotometric curve (what does it mean)?

A high 280 wavelength means that there is a high amount of purity in the sample 

How does a nanodrop give you DNA’s concentration/purity?

Concentration = ng/ul 

Purity = A260/280