(pt 1) exam #1 - intro to molecular diagnostics (cls 605)

what makes up a nucleotide? (3)

• pentose sugar

• nitrogenous base

• phosphate group (can have 1, 2, or 3)

  • free nucleotides in active form typically have 3 phosphate groups attached before they get attached to a DNA/RNA strand (triphosphates)

difference betwen RNA vs DNA nucleotide

RNA nucleotide has a OH group at the 2' carbon while a DNA nucleotide does not

• carbons are numbered 1'-5' with the nitrogenous based attached to carbon at the 1'

nucleoside

has no phosphate groups attached

• still has pentose sugar and nitrogenous base

nitrogenous bases

includes:

• Pyrimidines: cytosine and thymine/uracil (one ring)

• Purines: adenine + guanine (two rings)

how do nucleotides become nucleic acids?

• One strand of nucleic acid is generated when nucleotides attach to each other by their phosphate group

• This creates what is called the sugar-phosphate backbone of the molecule

  • bond is created via a condensation rxn

• bonding gives the molecule directionality--new nucleotides are added to the 3' end of a DNA strand

• ATTACHED VIA PHOSPHODIESTER BONDS

how does DNA become double stranded?

• Nitrogenous bases attach via HYDROGEN bonds

• Adenosine pairs with thymine

  • 2 hydrogen bonds

• Guanine pairs with cytosine

  • 3 hydrogen bonds

• For proper base pairing to occur the two DNA strands must run in opposite directions (antiparallel)

DNA packaging

• If DNA was not packaged in any way, the DNA from a single cell would be about 2 meters (6.6 ft) long

  • This would be approximately the equivalent of packing 24 miles of thread into a tennis ball

• packaged into a nucleosome made of 8 histone proteins

  • two of each: H2a, H2b, H3, H4

chromosome

threadlike structure of nucleic acids and protein found in the nucleus that carries genetic information (genes)

• most human cells contain 23 pairs of chromosomes (46 total)

chromatin

less condensed form of DNA and proteins; less organized compared to chromosomes

• found in interphase nuclei

euchromatin vs heterochromatin

• euchromatin: relaxed chromatin that is transcriptionally active

• heterochromatin: more condensed--no transcription (NOT active)

nucleosome

structural unit of a eukaryotic chromosome; consists of approx 150 bp of DNA wrapped around 8 histones

chromatid

one of two identical halves of a chromosome in preparation for cell division

human karyotype (general)

• Karyotype is the direct observation of metaphase chromosome structure

• Karyotyping can be used to see large changes in chromosomes such as:

  • Aneuploidy

  • Translocations

  • Large insertions or deletions

  • Inversions

karyotype presentation

• Cultured cells are arrested at metaphase

  • cells are most condensed and easiest to identify

  • Arrested cells are broken open

• Metaphase chromosomes are fixed and stained

• Chromosomes are digitally imaged through microscope

• Digital images of chromosomes are arranges to form a karyotype diagram

gene (definition)

units of information about heritable traits

• each gene has a particular locus

locus

specific spot/location on a chromosome

alleles

different forms of genes that arise through mutation

• A diploid cell contains 2 alleles at each locus

• Alleles on homologous chromosomes may be the same or different

exons vs introns

• exon: coding regions of a gene; included in the final mRNA transcript and translated into protein

• intron: non-coding sequences within a gene that are transcribed into RNA but are removed/SPLICED before translation into a protein

genotype vs phenotype

• genotype: genetic DNA composition of an organism

• phenotype: observable traits

dominant vs recessive allele

• dominant allele: allele that affects/masks the other allele with which it is paired

• recessive allele: allele whose effect is masked by the other allele with it is paired

homozygous vs heterozygous

• homozygous: genetically identical pair of alleles

• heterozygous: a pair of alleles for a trait that are not genetically identical

nucleic acid isolation (general)

• Goal: isolate DNA from inhibitors and impurities that can interfere with testing

  • direct interference: things like enzyme inhibitors

  • indirect interference: impurities that bind to DNA and make it unavailable for a reaction

samples that can be used for isolating nucleic acids

• Any sample containing the targeted nucleic acid

  • Whole blood / buffy coat

  • Bone marrow

  • Solid tissue

  • Lavage fluids

  • Bacteria, viruses, fungi

  • Organelles, mitochondria

best type of fixed specimens/fixatives for PCR

• Acetone

• 10% buffered neutral formalin

• FFPE specimens (formalin fixed, paraffin embedded)

mid/meh fixatives for PCR (not as good)

• Zambonis, clarkes

• Paraformaldehyde

• Formalin-alcohol-acetic acid

• Metharcan

less desirable fixatives for PCR

Carnoys, Zenkers, Bouins, B-5

general steps of DNA isolation (3)

1. Cell lysis

   • Disrupt membranes

   • Denature or degrade proteins

2. Extraction

   • Separate DNA from proteins and other impurities

   • Can use a solid or liquid phase

3. Precipitation

   • Further purifies DNA

   • Removes residual contaminants

(DNA isolation) cell lysis—disrupting membranes

• Detergents work by disrupting membranes

  • Ex: sodium dodecyl sulfate (SDS); cetyltrimethylammonium bromide (CTAB)

• Chaotropic salts work by disrupting hydrogen bonds

  • Ex: urea; guanidium hydrochloride

• Results in a "soup" of cellular debris and the contents of the cytoplasm

(DNA isolation) cell lysis—degrading proteins

• Proteases--enzymes that degrade protein

  • Work to:

    • Degrade proteins that bind DNA

    • Degrade nucleases (enzymes that degrade nucleic acids)

• Detergents and chaotropic salts

  • Denature proteins which:

    • Disrupts function

    • Makes more susceptible to proteases

(DNA isolation) alternative methods of cell lysis

• Sonication

• Freeze-thaw

• Physical disruption

• Manual grinding

(DNA isolation; extraction) liquid phase

• Phenol/chloroform--denatures proteins, doesn't mix with water so DNA in upper liquid phase (NOT preferred method)

• Procedure

  • Add phenol/chloroform to sample

  • Mix thoroughly to form emulsion

  • Separate phases by centrifugation

  • DNA in upper aqueous phase

  • Precipitated proteins rest on top of lower phenol chloroform layer

  • Upper layer containing DNA is pipetted into a new tube for further purification

disadvantages to liquid phase DNA extraction

• Lengthy

• Labor intensive

• Requires further purification

• Difficult to automate

• Safety issues

• Phenol chloroform carryover

(DNA isolation; extraction) solid phase

• DNA binds to silica (solid) in aqueous buffers w high salt concentrations (PREFERRED method)

• Procedure (what we did in lab)

  • Lyse cells in high salt buffer

  • Add silica beads OR pass lysate through a column with silica filter (DNA will stick to silica)

  • Wash off impurities with high salt buffer

  • Wash with alcohol to remove salt

  • Elute DNA (remove from solid) with low ionic strength aqueous solution

advantages to solid phase DNA extraction

• Generally DNA is pure enough so that further cleaning (precipitation) is not needed

• Quick, easy, safe, kit available (expensive though)

(DNA extraction) automated silica extraction

• Silica coated magnetic beads

• Increased binding kinetics/efficiency

• Enhanced removal of contaminants

• Commercially available

(DNA isolation steps) precipitation

• Add salt and alcohol to “clean” DNA

• Salt

  • Cations bind to phosphate groups of DNA and neutralize charge

  • Now DNA molecules won't repel each other

• Alcohol

  • DNA Is insoluble in alcohol

  • Adding alcohol makes DNA precipitate out of solution

  • Precipitated DNA is stringy and can be pelleted or "spooled"

modifications to DNA extraction method for RNA extraction

• RNA very susceptible to degradation

• Bench/equipment—keep separate, clean with RNase inhibitors

• Disposables—certified RNase free, rinsed in 0.1% diethyl pyrocarbonate (DEPC)

• Reagents—purchased RNase free or treated with DEPC, Trizol (guanidium, phenol, chloroform)

• Reactions—add RNase inhibitor

mRNA isolation

• Total RNA can be obtained with either:

  • A silica-based binding technique or

  • Purification procedure with TRIzol reagent

• From total RNA, mRNA purification is best accomplished via binding to Oligo dT fixed on a solid medium or column

methods for DNA quantification

• spectrophotometry

• fluorometry

• qPCR

• gel electrophoresis

methods for assessing purity of isolated DNA

spectrophotometry (purity)

methods for assessing quality of isolated DNA

gel electrophoresis

DNA quantification by spectrophotometry

• Absorbance at 260 nm—BASES absorb strongly at this wavelength

• An OD (optical density) of 1 corresponds to:

  • 50 ug/mL for dsDNA

  • 37 ug/mL for ssDNA

  • 40 ug/mL for ssRNA

  • 20 ug/mL for oligonucleotides

how to calculate concentration of DNA by spectrophotometry

conc of dsDNA (or whatever) in ug/mL = OD (at 260 nm) x 50 ug/mL x dilution factor

You are trying to find the concentration of your isolated RNA. You run 1 ul of sample on a spectrophotometer and the instrument returns an OD value of 0.5. What is your concentration?

0.5 x 40 ug/mL = 20 ug/mL

You have 100ul of isolated RNA of the concentration 20ug/ml. The protocol you need to run requires 5ug total of RNA. Do you have enough?

1. (20 ug/mL) / 1000 = 0.02 ug/ul

2. 0.02 ug/ul x 100 ul = 2 ug

3. no u don't have enough

DNA purity by spectrophotometry (280 nm)

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

• Ratio of 260/280 is a measure of purity

  • Pure DNA 260/280 ratio is 1.8 (range 1.6-2.0)

  • Pure RNA 260/280 ratio is 2.0 (range 1.8-2.2)

  • Contamination by protein or solvent (ethanol or phenol) results in low 260/280 ratios

• **If contaminated 260 nm readings cannot be used to estimate concentration

DNA purity by spectrophotometry (230 nm)

• Absorbance at 230 nm--peptide bonds, carbohydrates, phenol, thiocyanates, and other organics absorb at this wavelength

  • Ratio of 260/230 is a secondary measure of purity

    • Pure DNA ratio should be >1.6

    • Pure RNA ratio should be >1.8

    • Contamination results in low 260/230 ratios and may interfere with downstream applications

nanodrop (spectrophotometry)

• Sample size = 1-2 uL

• Quantitation range = 2 ng/uL tp >3 ug/uL

• No cuvettes

• Evaluates purity and quantity

DNA quantitation by fluorometry

• Mix DNA with a dye and then measure fluorescence—increased fluorescence is emitted when dye binds to dsDNA

• Hoechst 33258

  • Quantitate to 10 ng/mL

• Picogreen

  • Quantitate to 25 pg/mL (less sensitive to contaminants)

DNA quantitation by qPCR

• Compare Ct for specimen to standard curve for human genomic DNA of known concentration

• Amplify standard "housekeeping genes" such as

  • 18S rRNA

  • Beta Actin

  • G6PD

purity vs quality

• Purity: free from contamination

• Quality: is the DNA actually DNA and if the product is intact

what method assesses DNA quantity and quality?

electrophoresis

what method assesses DNA quantity and purity?

spectrophotometry

DNA quantification/quality by gel electrophoresis (general)

• DNA is negatively charged

• When placed in an electric field, DNA will migrate towards the positive pole (anode)

• An agarose gel is used to slow down the movement of DNA and separate by size

speed of DNA migration in gel electrophoresis depends on what?

• Strength of electric field

• Buffer

• Agarose density

• Size of DNA

  • Small DNA molecules move faster

basic equipment for gel electrophoresis

power supply, gel tank, cover, electrical leads, casting try, gel combs

(eletrophoresis steps/equipment) agarose preparation

• Agarose powder is mixed with a buffer and boiled until it's clear

• Agarose solution should be allowed to cool slightly and then poured into a casting tray

(eletrophoresis steps/equipment) gel preparation

• Gel combs are added before the gel solidifies, this creates wells where samples can be added

• When completely cooled, the agarose polymerizes which forms a flexible gel and the combs can be removed

• Add enough electrophoresis buffer to cover the gel so that it is submerged with at least 1mm of liquid covering the gel

• Make sure each well is filled with buffer

(eletrophoresis steps/equipment) sample preparation

• DNA sample is mixed with loading buffer—serves two purposes:

  • Contains glycerol: adds weight to the sample to help it sink to the bottom of the wells

  • Contains a tracking dye (bromophenol blue) so that you can monitor where the DNA is on the gel

(eletrophoresis steps/equipment) loading the gel

• Samples are loaded into wells

• Hold the pipette tip in the liquid right at the top of the well and expel the sample

• Be careful not to puncture the gel with the pipette

(eletrophoresis steps/equipment) running the gel

• Place cover on electrophoresis chamber, connecting the electrical leads to the power supply

  • Be sure the leads are attached correctly--DNA migrates toward the anode (red)

• When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber

• After the current is applied, make sure the gel is running in the correct direction

  • Bromophenol blue will run in the same direction as the DNA

(eletrophoresis steps/equipment) DNA ladder standard

contains fragments of DNA of known sizes so that you can determine the sizes of your DNA fragments

(eletrophoresis steps/equipment) staining the gel

• Ethidium bromide

• Binds to DNA, fluoresces under UV light

• Can be added before gel is poured or gel can be stained after running

• Ethidium bromide is a powerful mutagen!!!—NEVER EVER HANDLE WITHOUT GLOVES

(eletrophoresis steps/equipment) visualizing product (pic)

DNA quantity by gel electrophoresis

• Fluorescence intensity is proportional to the total mass of DNA

• Fluorescence of the sample is compared with DNA standards of known concentration

(electrophoresis practice example) You mix 2ul of your sample with 6 ul water and 2 ul loading dye and load the resulting mixture into lane 1

• What is the size of the product?

• What is the approximate concentration of your sample?

1. 1000-1500 bp (1250)

2. 40 ng/uL (take 80 ng and divide by 2)

   • only factor in the 2 uL of sample when deciding conc

DNA quality by gel electrophoresis

high quality nucleic acid appears as a tight band

RNA integrity number (RIN)

• assesses RNA quality throughout the electrophoretic profile, not just the 28S/18S ratio

• RIN > 8 = acceptable indicator of total RNA quality

• 28S/18S rRNA mass ratio is about 2.5

• A 28S/18S absorbance ratio > 2.1 is an indication that the total purified RNA is intact and HAS NOT degraded

human genome

• Current tally--21,000 protein coding genes

• Genes (25%)

  • 24% intron sequences

  • 1.1-1.4% exon sequences

• Intergenic sequences (75%)

  • 45% transposon-derived repeats (moveable gene regions)

  • 5% duplications

  • 3% repeats (ex: STRs)

  • 22% other (ex: spacer)

• 99.9% identity between individuals

• About 1 difference every 1250 bases between randomly selected haploid genomes

(human genome) sequence variation (general)

• Single nucleotide polymorphisms (SNPs) are the most common type of sequence variation

  • DNA chances involving 1 base pair

  • ~98% occur within non-coding sequences

  • ~2% occur in exons

SNPs vs mutations

• Mutation: any change in the "normal" human DNA sequence; mutation changes the sequence to something rare/abnormal

  • Single nucleotide changes ocurring in <1% of population = mutations

• Single nucleotide polymorphism: a DNA sequence variation that is common in the population; no single allele is regarded as the standard sequence

single nucleotide polymorphisms (SNPs)

• Over 100 million have been identified in the human genome

  • Some are common in the population with allelic frequencies of 0.1-0.5 (i.e. present in 10 to 50 of every 100 human genomes)

• On average, occur once every 1,000 bases in a person (~4 million in every person's genome)

  • Distributed throughout genome

  • Major cause of genetic diversity among different (normal) individuals, e.g. drug response, diseases susceptibility

  • Can detect using a variety of molecular methods

significance of SNPs

• Most do not change protein synthesis nor cause disease directly

• Few associated with disease (ex: sickle cell anemia)

• SNPs can serve as landmarks, since they may be physically close to a disease-associated gene on the chromosome

  • Because of this proximity, SNPs may be shared among groups of people with a disease (inherited as a haplotype)

haplotype

alleles that are close together on a chromosome and are usually inherited together

projects to understand human genetic variation

• HapMap project

• 1000 genomes project

disease causing mutations

• 70% SNPs

  • 49% missense

  • 11% nonsense

  • 9% splicing

  • <1% regulatory

• 23% small insertions/deletions

• 7% gross changes (large insertions/deletions, duplications, rearrangements etc)

• **point mutations do NOT always have a phenotypic effect

chart of genetic mutations

mutations that produce small/little changes

• silent/synonymous

• missense (conservative & non-conservative)

• splice mutations

• unstable trinucleotide repeats

• nonsense

• small insertions/deletions

• frameshift

silent/synonymous mutation

point mutation which does not change the amino acid

missense mutation

•  point mutation which results in a different amino acid being inserted

  • Conservative: similar amino acid is substituted for original (ex: hydrophobic AA for a hydrophobic AA)

  • Non-conservative: the changed amino acid is significantly different from the original which may affect protein structure/function

disease caused by missense mutations

• Hemoglobinopathies

• Cystic fibrosis

• Sickle cell anemia

unstable trinucleotide repeats

areas of DNA that have repetitive sequences are prone to expand or contract during DNA replication

• Diseases caused by unstable trinucleotide repeats:

  • Fragile X syndrome--(CGG)n in 5' UTR of FMR-1 gene

    • Normal: 5-44

    • Intermediate: 45-54

    • Premutation: 55-200

    • Full mutation: >200

  • Other examples: Huntington's disease, SCA (spinocerebellar ataxia)

splice mutations

• disruption of existing splice sites (intron is not removed from mRNA)

  • Creation of new splice site in exon

  • Ex: HbE missense mutation and splice error

nonsense mutation

point mutations which replaces a codon that codes for an amino acid with a stop codon

small insertion/deletion mutation

adds or removes bases; may or may not cause a frameshift

frameshift mutation

codon reading frame altered by insertion/deletion (occurs if the number of bases inserted or deleted is not a multiple of 3)

types of mutations that cause big changes (on chromosome level)

• duplication

• deletion

• inversion

• translocation

• nondisjunction

• isochromosome

duplication & deletion chromosomal mutation

• Duplication: gain of genetic information

  • Ex: MECP2 syndrome

• Deletion: loss of genetic information

  • Ex: Prader-Willi Syndrome

inversion chromsomal mutation

• no gain or loss of genetic information

  • Ex: hemophilia A (FVIII gene), Hunter syndrome (lysosome storage defect, IDS gene)

translocation chromosomal mutation

•  no gain or loss of information; breakage of chromosome and rejoining

  • Reciprocal or nonreciprocal

  • Ex: CML (t:(9:22)), AML

nondisjunction chromosomal mutation

• failure of homologous chromosomes/sister chromatids to separate properly during meiosis

  • Causes aneuploidy (too many or too few chromosomes)

isochromosome

• mis-division of the centromere results in duplication of one arm and loss of another

  • Found in some cancers

mitochondrial genome

• Circular chromosome

• Maternally inherited

• 16,569 bp; encodes 13 proteins

  • Involved in oxidative phosphorylation

• Mutations in mitochondrial DNA → disruptions in energy production

epigenetics

changes in gene expressions that do not involve changes to nucleotide sequence; can be heritable changes in some cases

two major epigenetic processes

• Chromosome structure changes

  • Affects gene availability (ex: histone modification)

• DNA methylation

  • Can block transcription

DNA methylation

• Primarily cytosine methylation in the context of CpG dinucleotides (islands)

• CpG islands are stretches of DNA (500-1500 bp) with more than 60% GC

• Found at promoters and contain the 5' end of the transcript

(epigenetics) transcriptional silencing

hypermethylation of DNA in the promoter region can inhibit gene transcription

(epigenetics) transcriptional activation

hypomethylation of DNA in the promoter region can activate gene transcription

(epigenetics) genomic imprinting

• one of the alleles of a gene is silenced (no transcription), depending on the parent of origin

  • X inactivation--one of the X chromosomes in individuals with two X chromosomes is inactivated by methylation (no transcription)

restriction enzymes

• In bacteria, part of protection from bacteriophages

• Cleave nucleic acid at specific nucleotide sequences

• Bacteria protect their own DNA by methylation at sequence recognized by the restriction enzyme

• Are thousands of restriction enzymes:

  • AluI - AG↓CT - Arthrobacter luteus

  • BamHI - G↓GATCC - Bacillus amyloliquefaciens H

  • EcoRI - G↓AATTC - Escherichia coli RY13

  • HindIII - A↓AGCTT - Haemophilus influenzae Rd