DNA structure, Organization, Purification, Quantification

Karyotype preparation

* cultured cells are arrested at metaphase because this is when the cells are most condensed and easiest to ID. The arrested cells are broken open * metaphase chromosomes are fixed and stained * chromosomes are digitally imaged through microscope * digital images of chromosomes are arranged (by size) to form a karyotype diagram

Modifications of disposables for RNA extraction

- Certified RNase free OR - Rinsed in 0.1% diethyl pyrocarbonate (DEPC)

What are the best fixatives for DNA extraction by PCR?

- acetone - 10% buffered neutral formalin - FFPE (formalin fixed, paraffin embedded)

Liquid phase extraction procedure

- add phenol/chloroform to DNA dissolved in water - mix thoroughly to form an emulsion - separate phases by centrifugation - DNA remains in upper (less dense) aqueous phase - precipitated proteins rest on top of the lower phenol/chloroform layer - upper layer is removed and DNA precipitated

Modifications of reagents for RNA extraction

- certified RNase free OR - add 0.05-0.1% DEPC (except in Tris-based solutions which inactivate DEPC) - Trizol (guanidinium, phenol, chloroform)

Solid phase DNA extraction procedure

- lyse cells in high salt buffer - add silica beads to lysate, or pass lysate through a column containing a silica filter - wash impurities off with a high salt buffer (to keep DNA bound to silica) - Wash with alcohol to remove salts and other impurities - elute DNA off silica with low ionic strength aqueous solution DNA is generally sufficiently pure so that further cleaning (precip) is not needed.

total RNA isolation can be obtained by:

- silica based binding technique - precipitation procedure with TRIzol reagent (phenol and guanidine isothiocyanate reagent)

NA isolation alternatives

- sonication - physical disruption with beads (bead beating)

DNA isolation common steps (3)

1. cell lysis - disrupt membranes - denature/degrade proteins 2. extraction - liquid or solid phase - pulls out impurities 3. precipitation - further purifies the DNA

What are the main ways to lyse a cell?

1. detergents 2. chaotropic salts 3. proteases

what are the 2 methods of nucleic acid isolation?

1. direct interference - enzyme inhibitors 2. Indirect interference - impurities bind the DNA and effectively make it unavilable for enzymatic reaction

What are the 2 types of functional DNA sequences?

1. families of coding genes (and related pseudogenes) 2. noncoding functional sequences (telomeres- no function of making proteins but protects as its function)

What are the types of non-functional repetitive DNA?

1. repeats in centromeric heterochromatin (basically just centromeres) 2. variable number tandem repeats (used in forensics) 3. transposed sequences (transposons or retrotransposons)

DNA isolation: MagnaPure

1. sample 2. add lysis buffer and proteinase k 3. NA binding to magnetic particles (DNA or RNA is removed by DNase/RNase) 4. Magnetic separation 5. removal of cellular debris by washing step 6. magnetic separation of the NA bead complex 7. NA elution at high temps during removal of beads

Eukaryotic DNA can be broken down into 3 types

1. single-copy functional genes (only 1 copy) 2. repetitive DNA 3. spacer DNA (not either above)

an OD of 1 corresponds to ___________ for oligonucleotides

20 µL/mL

what ratio is the second measure of purity?

260/230

What wavelength is used to determine purity of NA for spectrophotometry? why?

280nm - peptide bonds and some organic solvents absorb strongly at this wavelength

an OD of 1 corresponds to ________ ssDNA

37 µg/mL

an OD of 1 corresponds to _____ RNA

40 µg/mL

DNA polymerase adds nucleotides in what direction?

5' to 3'

an OD of 1 corresponds to _________ dsDNA

50 µg/mL

What should the 260/230 ratio be for pure DNA?

>1.6

What should the 260/230 ratio be for pure RNA?

>1.8

Which bases are pyrimidines?

Cytosine and Thymine

DNA is __________ and ______________

DNA is complementary and anti-parallel

What is the structural difference between DNA and RNA?

DNA is deoxy- meaning there is no hydroxyl (OH) group at the 2nd carbon in the nucleotide. RNA has a hydroxyl group located at the second carbon

Electrophoresis

DNA is negatively charged, so when placed in an electrical field, the DNA will migrate toward the positive pole (anode) - the agarose gel is used to slow the movement of DNA and separate it by size.

Heterochromatin

DNA that is densely packed around histones. The genes in heterochromatin are generally inaccessible to enzymes and are turned off. - inactive and not highly transcribed

nucleosome structure

DNA wrapped around 8 histones, sealed with another histone

Explain how DNA is condensed

DNA wraps around histone proteins to form nucleosomes. The nucleosomes will coil and stack to form fibers (known as chromatin). Chromatin will loop and fold (with the help of other proteins) to create chromosomes

What is the purpose of a DNA ladder?

Makes it easy to determine the sizes of unknown DNAs.

sample preparation for gel electrophoresis

Mix the samples of DNA with DNA sample loading buffer (w/ tracking dye to allow samples to be seen and have density).

Liquid phase extraction is done by what chemicals?

Oldest method and still being used Phenol/chloroform - denatures and extracts proteins - non-miscible with water

mRNA purification is best accomplished via binding to ____________ on a solid medium or column

Oligo dT fixed

Heterozygous

Pair of alleles for a trait are not genetically identical

Running the gel

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply (making sure they are attached correctly). When turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Why is DNA consdensed?

Protection

Which bases have two rings?

Purines: - Adenine and Guanine

How does fluorometry work

Quantitates NA by mixing DNA with a dye and then measuring fluorescence in a fluorometer

Agilent bioanalyzer

Quantitation and Quality for RNA Gel electrophoresis in a nanofluitic chamber - separates based on size - laster at bottom that measures fluorescence

Pyrophosphate

Released during the addition of deoxyribonucleotides to the nascent DNA chain

What is the goal of nucleic acid isolation?

Remove inhibitors and impurities that interfere with testing

automated silica extraction

Silica-coated magnetic beads - proprietary beads - increased binding kinetics/efficiency - enhanced removal of contaminants - commercially available

DNA migrates towards the _________ a. anode b. cathode

a. anode (red)

Specimens for NA extraction

any sample containing nucleic acid - whole blood - buffy coat - bone marrow - solid tissue - lavage fluids - bacteria, viruses, fungi - organelles, mitochondria

the movement of DNA in gel electrophoresis is __________ a. linear b. exponential

b. exponential

which statement is true? a. spectrophotometry can accurately determine concentration, purity, and quality of NA b. spectrophotometry can accurately determine concentration, purity, but not quality of NA c. spectrophotometry cannot accurately determine concentration, but can determine purity and quality of NA

b. spectrophotometry can accurately determine concentration, purity, but not quality of NA

Concentration calculation

concentration= (OD) (constant)(dilution factor)

What does a high 160/180 ratio indicate?

contamination by RNA - use RNases and then do a precip step

What does a low 160/180 ratio indicate?

contamination by protein or solvent - re-do phenol and chloroform step to remove proteins

What method is used to determine if NA is intact?

electrophoresis - degraded NA will absorb as strongly at 260nm as non-degraded sample

Staining the gel

ethidium bromide binds to DNA (sits between the base pairs) and fluoresces under UV light, allowing the visualization of DNA on a gel * can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

What is included in the loading buffer?

glycerol - for weight so sample will sink into well bromophenol blue - to see the sample (doesnt selectively bind NA) - used to monitor extent of electrophoresis

a sugar and base form _______________, when phosphorylated it is made into ______________

nucleoside, nucleotide

What is the building blocks of nucleic acid?

nucleotides

what is absorbed strongly at 230nm?

peptide bonds, carbohydrates, phenol, thiocyanates, and other organics

what causes a low 260/230 ratio?

protein or solvent (ethanol or phenol)

160/180 ratio for pure DNA and pure RNA

pure DNA: 1.6-2.0 pure RNA: 1.8-2.2

the ratio of 260/280 absorbance is a measure of ________-

purity

What does the nanodrop reading evaluate?

purity and quantity

What does fluorometry evaluate?

quantitation

________ are the markers for RNA quality in a prep

rRNA

What 2 chemicals are added for precipitation?

salt - cations bind to the phosphate groups of DNA molecules to neutralize the negative charge - DNA molecules can clump instead of repelling each other when alcohol is added alcohol - DNA is insoluble in alcohol - adding alcohol makes the DNA clump together and precipitate out of a solution - precipitated DNA appears as fluffy, stringy, web-like threads thats can be pelleted or "spooled"

Spectrophotometry: Nanodrop

sample size: 1-2 µL quantitation: 2ng/µL - >3 ng/ul no cuvettes- sample is applied directly onto optical head

Solid phase DNA extraction uses?

silica beads

What does the speed of migration for gel electrophoresis depend on?

strength of the electrical field, buffer, density of agarose gel size of DNA - small DNA moves faster than large DNA molecules

If the 160/180 ratio is contaminated, what do you have to do for the 160 reading?

the 160 reading cannot be used and must be decontaminated and re-done

loading the gel

the loaded pipette tip is placed over a well and the DNA/loading buffer mixture is gently expelled. The sample should sink inot the well but be careful not to puncture the gel with the pipette tip.

how many hydrogen bonds are between C and G?

three

How many hydrogen bonds are between A and T?

two

Two different dyes for fluormetry

Hoechst 33258 - quantitate to 10 ng/mL Picogreen - quantitate to 25 pg/mL, and less sensitive to the presence of contaminants

Modifications of bench/equipment for RNA extraction

Ideally, a separate lab area or separate lab equipment designed RNase free - clean with RNase inhibitors

Quality by Electrophoresis

High quality NA appears as a tight band during electrophoresis * note on the RNA electrophoresis, you cannot differentiate mRNA*

modifications of reactions for RNA extraction

Add Rnasin (promega)

Which bases are purines?

Adenine and Guanine

What are the 4 bases of DNA?

Adenine, Thymine, Guanine, Cytosine

Gel preparation

After the agarose has cooled, it will polymerize and form a flexible gel. The combs and tape should be removed. Electrophoresis buffer is added to cover the gel to a depth of about 1 mm. Each well needs to b filled with buffer.

How do you prep agarose?

Agarose is insoluble at room temperature, so the solution is boiled until it is clear. After boiling, it must be cooled and then it can be poured into the casting tray (try to avoid air bubbles)

Dominant

An allele that affects/masks the other allele with which it is paired... results in observed phenotype

Recessive

An allele whose effect is masked by the other allele with which it is paired

why do we measure concentration at 260nm for NA?

Bases absorb strongly at this wavelength

Quantitation by qPCR

Compare Ct for specimen to standard curve for human genomic DNA of known concentration amplify standard "housekeeping genes: such as - 18s - beta actin - glucose-6-phosphate dehydrogenase

When lysing the cell, what degrates and denatures proteins?

Degradation (chopping) - proteases destroy nuclear proteins that bind to DNA and cytoplasmic enzymes that breakdown and destroy DNA - Proteinase K is the most commonly used protease because it is not denatured by SDS or Chaotropic salts Denaturation (unfolding) - done by detergents and chaotropic salts - makes them more susceptible to proteases

When lysing the cell, what disrupts the cell membrane?

Detergents - SDS* - CTAB Chaotropic Salts - urea - guanidinium hydrochloride*

What are the 2 types of families when it comes to coding genes?

Dispersed gene families Tandem gene families

Why is DNA spiral?

Due to stoichiometry and negative charged phosphates that repel each other. Bases are stacked to be ontop of each other

Why is DNA antiparallel?

Each chain has a 5' end and a 3' end & they run in opposite directions.

Quantitation by Electrophoresis/Fluorescence

Fluorescence intensity is proportional to the total mass of DNA - fluorescence of the sample is compared with that of DNA standards of known concentration to estimate the concentration of the sample.

What are the 2 types of repetitive DNA?

Functional and non-functional

What system is best for determining quality of NA?

Gel electrophoresis

genes vs alleles

Genes: - units of information about heritable traits - distributed among chromosomes (eukaryotes) - each gene has a particular locus Alleles: - different forms of a gene. - arise through mutation - diploid cell will have a pair at each locus - can be homogenous or heterozygous

Homozygous

Genetically identical pair of alleles

Genotype

The combination of alleles in a genome (genetic potential)

Euchromatin

The less condensed form of eukaryotic chromatin that is available for transcription (genetically active)

Phenotype

The observable traits

True or false: only mRNA has a poly A tail out of all RNA

True