Molecular genetics tech quiz 1

Dna structure

Phophate backbone

deoxyribose sugar

nitrogenous base

DNA ladder

side rails phosphate and sugar

rings nucleotides held together by H bonds

Chargaffs basepair rule

AT

GC

pyrimidine purine

Buccal swab

swab inside of cheeks.

saliva has WBC increases DNA content

place swab into microcentrifuge tube containing cell lysis solution

Organic extraction methods

Uses organic chemicals, phenol, chloroform

Inorganic methods

uses inorganic chemicals, detergents, ethylenediamine tetraacetic acid (EDTA), acetic acid, salt (salting out spooling)

Solid phase

DNA immobilized on solid support, beads, or columns.

Organic DNA ISO

Lysis NaOH, SDS (sds to solubilize cell membrane NaOH breaksdown cell wall.

acidification (acetic acid, salt)

Extraction (Phenol, chloroform)

DNA Precipitation (ethanol)

Extraction separates

into 2 phases

organic phase

Lipids and proteins

Aqueous phase

nucleic acids

Phenol/Chloroform/Isoamyl alcohol ratio

25:24:1

Phenol pH 7-8

removes proteins and other contaminating materials from aq DNA solutionC

Chloroform

Denatures proteins and removes residual phenol

Isoamyl alcohol

To reduce foaming

Final aqueous phase

must be free from phenol and protein contamination

Nucleic acids precipitated

with ethanol or monovalent cations (sodium acetate) near 0c

phenol chloroform extraction

DNA sample → Phenol chloroform → centrifugation → layers (aqueous interphase organic) aqueous separated and moved into new tube→ isopropanol →centrifugation → DNA pellet left.

Inorganic extractions

alternative to organic extractions

reduces exposure to hazardous reagents

commercial kits available

Inorganic extraction kits

Salt precipitation

absorption to silica surface

anion exchange chromatography

In silica surface

GITC included to inhibit nucleases and promote binding of nucleic acids to silica beads/ membranes

elute nucleic acids from beads/membranes after washes with low salt aqueous solution

safely and quickly produces high quality high purity nucleic acid preparations

Inorganic DNA isolation (salting out)

lysis (tris, EDTA, SDS, Prot.K, heat) → protein precipitation (sodium acetate) → DNA precipitation (isopropanol)

(sodium acetate - precipitates membrane, C, H, O, Lipids, proteins, and monomers.)

Lysis buffer

100mM Tris HCl pH 8.5

0.5M EDTA

10% SDS

5M NaCl

20mg/ml proteinase K

100mM tris hcl ph 8.5

tris or trix (hydroxymethyl) aminomethane

used as ph buffer

ideal characteristics

not hydroscopic

easily dissolved in water, available in high purity, does not precipitate calcium salts, stable in solution at room temp for month, does not appear to inhibit many enzymes

0.5M EDTA

EDTA (Ethylenediamine tetracetic acid) weakens the cell by binding the divalent cations (Mg++ and Ca++) needed for membrane stability. Breaks cells open

inhibits nucleases released from cells

10% SDS

SDS (sodium dodecyl sulfate) biological detergent causes cell mem to breakdown further. Emulsifies lipids and proteins disrupting polar interactions holding membrane together. Detergent forms complexes with lipids and proteins causing them to precipitate out of solution

Major ingredient in laundry detergent

dissolve cell membrane, denature proteins

5M NaCl

NaCl enables nucleic acids to precipitate out of alcohol solution. Shields negative phosphate end of DNA

20mg/ml proteinase K

enzyme that degrades proteins

non specific serine protease used to purify target material like DNA from contaminating proteins and to inactive other enzymatic activities 55 c

Protein precipitation

sodium acetate causes lysed cell debris to precipitate and separate from DNA.

centrifuge tubes to protein precipitate will form pellet

DNA in solution

pour DNA solution into new tube containing alcohol

Causes dna to condense to make small white puff

after centrifugation DNA alcohol mix DNA will precipitate to form a small white pellet.

What to do with pellet

Isopropanol is added to DNA lysate and samples mixed until precipitation complete 50 inversions.

wash dry

resuspend pellet in PCR grade water or TE buffer

incubate 65 C for 30-60 min or RT overnight

store dna at 2-8c

Solid phase DNA ISO

Lysis (supplied reagents) → Acidification (Supplied reagents) → Adsorption (Low pH) → wash (supplied buffer) → Elute DNA (Low salt).

Common denominator in lysis

Chaotropic salt, forms salt bridges that disrupt nucleic acid association with H2O

Solid phase/affinity method

Bind DNA to solid support (silica) spin columns that contains silica based gel that can bild DNA in high salt

Wash off nucleic acids (elute) use DI water or low salt buffer

Advantage of solid phase

fast bases for automated extraction systems

Disadvantage of Solid phase

works only with small sample sizes

important factors isolating DNA

quality purity concentration contaminants proteins lipopolysaccharides tissue preservatives

optimize important factors by selecting proper sample source and technique.

nucleic acid storage

prevent enzymatic or physical damage.

chelating agents prevents cation damage EDTA

Chaotropic agents denaters RNase and DNase GITC

refrigeration reduces enzyme activity

Determine storage conditions genomic DNA

short term TE buffer 4 C

long term in ethanol ± salt at 20-80 C

PCR grade dna storage

Working aliquot short time storage 4 C

all pcr product not being used should be stored 20 C immediately

Aliquot

should not be repeatedly frozen and thawed.

spectrophotometry

when light travels through matter it slows down

Refactive index

ratio fo speed of light in space to speed of light in substance

optical density

degree to which refractive medium slows transmitted rays of light (absorbance)

Lambert beets law

A=E*I*c

E= molar absorptivity constant 0.02ug/ml

if graph of absorbance versus concentration, if absorbing species obeys Lambert-Beers Law, you will get a straight line

230nm

detection of protein based peptide bonds

260 nm

detection of nucleic acids DNA and RNA

280 nm

detection of proteins based upon presence of aromatic aa

320 nm

background correction (particulates in solution or dirty cuvettes

spectrophotometer

measure nucleic acid and protein.

purity = A260/A280

ratio 1.6-2.0 is pure

ratio < 1.6 indicated protein or organic contamination

if greater than 2.0 RNA contamination

concentration

OD260 1 corresponds to 50 ug/ml dsDNA

OD260 1

40ug/ml ssRNA

OD260 1

33ug/mL SSdna

Dna concentration

(a260-a320) x dilution x 50ng/ul = ng/ul

total DNA

DNA conc x final volume = ng

purity

(a260-a230)/(a280-a320) = 1.6-2 acceptable range.

RNA concentration

(a260-a320) x dilution x 40ng/ul = ng/ul

total rna

rna conc x final vol = ng

purity

(a260-a230)/(a280-320)= 2-2.2

wavelength nucleic acid measured

maximum 260 nm

DNA RNA not discriminated

Will protein contaminants cause discrepancies

yes can absorb same wl but peaks at a230

bioanalyzer

measure quantity and quality

gel electrophoresis

gdna high molecular weight will migrate low mobility

measures quality and quantity.

only quantity if run against genomic ladder.

Nucleic acids

quantitation of NA

UV absorbance heoscht 33258 ethidium bromide picogreen

how to choose method

accuracy ease of use sensitivity lack of interfering substances purity of sample.

Ethidium bromide

Can be used to quantify both DNA and rna

flat planar structure.

needs double stranded structures.

less sensitive to ssdna and rna.

not significantly impaired by high GC content

excitation at 546 nm

20 fold less sensitive than hoescht 33258 dye

disadvantage carcinogenic/toxic.

what is PCR

amplifies segment of DNA

segment represents small part of large complex micture of DNA

can be thought of as molecular photocopier

PCR is used

to copy DNA in tube

DNA produced by PCR

used for sequencing, DNA cloning, southern and northern hybridization

why amplify DNA why not use directly after isolation?

starting material is limited and multiple testing. For genetic disease only small part of DNA needs investigation. PCR helps amplify copy.

how powerful is PCR

Can amplify DNA in 1 hr

template DNA not needed highly purified

product can be digested with re seq and cloned

PCR can amplify little amount of dna

components of PCR mixture

template DNA, primers, dNTPs, Taq dNA poly, buffer sol, divalent cations, sterile DI water.

reaction buffer

includes tris ammonium pattasium ions serum albumin

keeps master mix at proper ph so PCR reac take place

Catalyst

Mg2+

stored at room temp

nucleotides dNTPs

eq amounts of each nucleotide added to mix stored at -20c

primers

both specific and universal primers can be used

stored at -20c

dna poly

typically thermal stable Taq stored at -20c

reaction set up

C1V1=C2V2

master mix

h20 mg2+ buffer Taq poly dNTP

pcr primers

ss 18-30 base dna frag complementary seq

primers determine specificity of PCR

distance between primer and binding sites plus primers determine size of product

primer design

sequence avoids 3 or more G or C near 3’end

GC content 40-60%

avoid A or T at 3’ end

avoid mismatches at 3” end

avoid complementary seq within primer and between

performing PCR

contains all comp for DNA synth

analyze PCR

subject reaction mix to amplification program

PCR cycle

denature 90-96c 30 sec

annealing 50-68c 30 sec

extension 70-75c 30-45 sec

pcr phases

lag exponential linear and plateau

2^N

plateu occurs when

reagents depleted products re-anneal polymerase damaged unwanted products accumulate

increasing cycle number above 35 little positives

thermal cylcers

pcr cyclers available from many suppliers

block and multi block

reactions in tubers or 96 well micro-titer plate

thermostable polymerases

used to withstan repeated high denaturation temps

To know no contamination

no template control

PCR grade water used instead of DNA template

Positive control needed in PCR

dna sample rigorously tested and adequately optimized for given set of primers.