Molecular Diagnostic Exam 3 Study Guide

Chromosome mutations

-affect structures of entire chromosomes -require movement of large chromosomal regions either within the same chromosome or to another

What is a microtome?

-instrument used to cutting thin sections of tissue or material to be visualized under a microscope

How many loci are used in STR testing by the FBI?

13 STR loci used in identification of individuals in US identification of STRs is powerful tool in forensics number of repeats within an STR is referred to as an allele

What percentage of the genome codes for proteins?

2% genetic variations occurring in more than 1% of population is considered useful polymorphisms for genetic linkage analysis more frequently polymorphism is present in people with disease phenotype, more likely an affected gene is located close to polymorphism, these genes are inherited

What is the purpose of identifying AMEL?

AMEL locus (amelogenin) is used to identify male individuals, Y allele of AMEL gene is 6 bp larger than X allele amplification and electrophoresis reveals 2 bands or peaks for males (XY) and one band or peak for females (XX) female DNA can be in abundance so using Y specific primers in Y-STR can be amplified by PCR from male female mixtures

What is ASO?

Allele-specific oligomer hybridization (ASO) -utilized differences in melting temperatures of short sequences of 20bp with 1 or 2 mismatches and those with no mismatches -synthetic ss-probes with normal or mutant target DNA sequence are used -at specific annealing temperatures, probe will not bind to near complementary target sequence with 1 or 2 mismatched bases, whereas probe with perfect complementary sequence will bind -probes are immobilized on membrane mostly used to identify frequently occurring mutations of well-characterized genes, ex. BRCA1, CF patients use dot-blot analysis

Calculate CPI

CPI: product of all PI of each loci tested, multiple the PIs to get big CPI number PI: likelihood of paternity, calculated for each locus in which both father and child share an allele, how many more times likely child's allele is inherited from alleged father than by another man, frequent allele in population has low PI and vice versa

Identify electropherograms with dye blobs

DNA contamination: presence of dye blobs: flashes of fluorescence that show as large broad peaks combination of short and large peaks indicates low DNA concentration and contamination occur when Sanger reaction products are not cleaned up properly for automated sanger

False negative and false positives

False negatives: nucleic acids degraded or inhibited, organisms may be present, prevent this by doing proper collection and transport and remove inhibitory substances False positives: contamination, carryover, dead organism in sample

Differentiate between homologous extrinsic, heterologous extrinsic, and heterologous intrinsic controls

Homologous extrinsic: target derived control with non target derived sequence, ensure correct extraction and amplification Heterologous extrinsic: non target derived control, added before nucleic acid extraction, uses second set of primers Heterologous intrinsic: nontarget sequences naturally present in sample

Differentiate between intragel and intergel precision. genotyping considerations

Intragel precision: comparing bands in the same gel Intergel precision: comparing bands between separate gels

Differentiate between kinship index and avuncular testing

Kinship index: sibling testing Avuncular testing: for aunt/uncles or niece/nephew

Differentiate between linkage equilibrium and linkage disequilibrium

Linkage equilibrium: when 2 or more alleles occur randomly in population Linkage disequilibrium: when alleles do not occur randomly, if allele or locus is always present in affected family, that locus must be closely linked to gene responsible to the phenotype/disease

Explain how molecules are separated using MALDI-TOF and describe their migration pattern

MALDI: Matrix-assisted laser desorption/ionization -produces ions by firing laser pulse into sample coated with matrix -ions fly to the detector at speed based on its mass and charge (MALDI-TOF) -ions separate according to their mass/charge ratio so that lighter ions travel faster and reach detector before heavier ions do

What is MCA?

MCA: melt-curve analysis -similar to ASO, exploits sequence and stacking directed denaturation characteristics of DNA duplexes -post-amplification step of RT-PCR -sequence differences result in different melting characteristics and melting temperatures (TM) -(TM) illustrated as a peak, specimens with identical sequences should yield overlying peaks at expected ™, whereas specimens continuing different sequences will yield 2 or more peaks at different temperatures

Differentiate between reagent blank and amplification control used for ID of microbes

Reagent blank: contamination control Amplification control: aimed at a target that is always present -housekeeping genes like groEL, rpoA, or for eukaryotes beta actin of G3P

SSCP. What is a conformer?

SSCP: Single-stranded conformation polymorphisms -based on preference of DNA to exist in ds state -in absence of complementary strand, DNA forms intrastrand duplexes to attain as much ds condition as possible -each folded strand forms 3D structure- conformer, shape depends on complementary nucleotides available for hydrogen bonding and folding, single base difference in DNA sequence can cause conformer to fold differently -Dilute concentration of PCR products from sample -Denature samples: NaOH, formamide, and heat -Resolve in polyacrylamide gel -Bands are analyzed, patterns different from normal sequence control conformers indicate presence of gene mutations

What are the characteristics of proper selection of sequencing targets for id of microbes?

accuracy of test is limited by choice of target sequences for primer or probe hybridization primers should target sequences unique to microorganism of interest ???

Gene mutations

affect single genes, small changes in DNA sequences Substitution: nucleotide is replaced by different one Frameshift: insertions of deletions Insertions: extra nucleotide is added to existing sequence Deletions: nucleotide is removed from sequence

What is stutter? genotyping considerations

errors due to pol missing a repeat during the replication process resulting in 2 or more different species in amplified product- extra bands or peaks

Calculate overall frequency

the more loci tests, the higher the probability that the locus genotype positively identifies an individual- match probability product rule: frequency of a set of alleles or genotype in population is product of the frequency of each allele separately OF = F1 x F2 x F3... OF is overall frequency and F represents frequency of each individual allele in population OF for D (1 of 10 people) and E (1 of 50 people), OF = 1/10 x 1/50 = 1/500 = 1 in every 500 individuals

Identify electropherograms with too much DNA

too much template is shown by high intensity peaks in beginning of run, then drops off quickly

Indirect ELISA

-2 step process for detection -advantages: high flexibility- same secondary antibody may be used for several primary antibodies -disadvantages: long protocol, potential cross reactivity from secondary antibody antigen is coated onto wells by passive adsorption -primary antibody specific for antigen binds to the target -labeled secondary antibody against host species of primary antibody binds to primary antibody for detection -substrate is added and color/signal develops

Biochemical methods used in the detection of gene mutations

-Biochemical methods are used to analyze the change in protein structure of function rather than search for potential mutations since you can easily get lose in the thousands of nucleotides -Enzyme immunoassay: hormones, drugs, use of antibodies specific to certain antigen, cancer biomarkers, other metabolites- blood, tissue, urine -Immunohistochemistry: detects protein abnormalities in situ -High performance liquid chromatography: can analyze multiple proteins at once- proteomics -Gas chromatography -Mass spectrometry: can analyze multiple proteins at once- proteomics

Nucleic acid analyses used to identify gene mutations

-Hybridization based (FISH/Southern blots) -Sequencing (polymerization) based -Enzymatic and chemical cleavage -Nextgen sequencing Nucleic acid analyses: -detection of inherited mutations, use noninvasive specimen (blood) -detection of somatic mutations more challenging as cells harboring mutation may be only small fraction of total specimen- amplification PCR -biochemical methods do not indicate type of mutation (silent, conservative, or nonconservative)

Gas chromatography

-analytical technique used to detect components of sample mixture to determine the quantities of them as well as separating them -sample is introduced to column and vaporized into a gas, strength of interaction or dissolution of sample components into liquid phase will result in varying retention times in column -effluent (once separated) from column is read by detector, such as flame ionization detector -used for detection of drugs Mobile phase: inert gas, helium or nitrogen Stationary phase: high boiling point liquid that is absorbed to an inert solid support in column

Missense mutations

-change in nucleotide results in encoding for a different amino acid -severity of the mutation depends on amino acid substitution and location within the polypeptide Conservative: change amino acid for one with similar biochemical properties, may affect function, ex. alanine to glycine? Nonconservative: change amino acid for one with completely different biochemical profile -insertions or deletions cause this

Genome mutations

-changes in the number of chromosomes Euploid: cell/cell population with normal complement of chromosomes Aneuploid: increased number of chromosomes -can result when there are more than 2 copies of one or more chromosomes -down syndrome: 3 copies of chromosome 21 -loss of whole chromosomes is not compatible with survival of organism

Mass spectrometry

-converts molecules to ions that can be moved in a magnetic field based on their charge and mass -ion source sends high energy electrons that hit target sample molecules separating them into ions -collection of particles is accelerated and focused into a beam through a magnetic field that deflects the ions according to their mass and charge -ions are aimed at detector and readout of instrument is a spectrum X axis: mass/charge value Y axis: abundance of ion -molecules in sample are identified by their characteristic spectrum of peaks compared to database

Next-Gen Sequences

-massive parallel sequencing -designed to sequence large number of templates carrying millions of bp simultaneously in run that takes few hours, produces raw data, bioinformatics

Sandwich ELISA

-most commonly used, used to identify and quantity antigen concentration in samples -advantages: more sensitive than direct and indirect, gives fast/precise results of antigens in sample, high specificity: 2 antibodies detecting different epitopes on same antigen, high flexibility and sensitivity: both direct and indirect methods can be used -disadvantages: demanding design- finding 2 antibodies against same target that recognize different epitopes and work well together is tricky -one antibody is coated on surface of well plate and used as a capture antibody to facilitate immobilization of the antigen, non specific binding site blocked and antigen is then added -other antibody is conjugated and facilitated the detection of the antigen, the detection antibody is added -enzyme conjugated detection reagent added -substrate is added and color/signal develops

Nonsense mutations

-nucleotide change codes for a stop codon which halts polypeptide translation -insertions or deletions cause this

Immunohistochemistry (IHC)

-performed on thin slices of fixed tissue, fixation of tissue in formalin preserves tissue morphology -section from fixed tissue embedded in paraffin are made on microtome antigen retrieval: enzyme digestion of tissues with protein digesting enzymes (proteinase K, trypsin, chymotrypsin) or by heating or using buffers can uncover antigen epitopes for EIH antibodies to bind -frozen tissues can be used -substances can interfere with binding or cause background which makes interpretation of results difficult endogenous peroxidases, fluorescence, nonspecific antibodies in tissue -preventing background staining: hydrogen peroxide, UV light, 1% serum blocking solution: serum proteins (aluminum or casein) and detergent (tween 20) will minimize nonspecific binding by preventing primary antibodies from non specific binding -washing steps -use correct antibody dilutions -use counterstains after addition of secondary antibodies -run parallel controls

Explain Maxam-Gilbert sequencing

-required ds or ss version of DNA region to be sequenced with one end radioactively labeled -labeled template put into 4 tubes, each tube was treated with different chemical with or without high salt, addition of strong reducing agent, 10% piperidine, ss DNA would break at specific nucleotides -fragments then resolved by electrophoresis -sequence was inferred from bands on film -lane in which that band appeared identified nucleotide, reading from bottom of gel to top

illumina sequencing

-sequencing by synthesis technology -multiple instruments -Next-gen sequencing by synthesis- combines bridge amplification and sequencing by synthesis

What is an electropherogram? Identify an electropherogram for a properly performed Sanger sequencing run. How long are the DNA fragments that can be sequenced by Sanger methodology? What is indicated by the Q-value?

-series of peaks, one for each of the 4 fluorescent dyes, software assigns one of 4 colors associated with each of the fluorescent dyes and text letter to peak for ease of interpretation -it is a chart of light emission over time, Y axis-intensity, X axis scan numbers, used to sequence 400-800 bp -want sequence in middle highest confidence that signal indicates that bp -base calling: process of identification of bases in sequence by sequencing software, when base call not clear, letter N will replace nucleotide but does not mean it won't be used it just cant be identified -beginning of chart: messy peaks, sequences before 30 pb not reliable, gray bars indicate Q value -Q value: indicates probability of correct call; anything greater than 20 is acceptable -Middle of chart: peaks begin to decline, data still reliable, sequence stops where dye terminators are used so longer the sequence the less of it there will be so peaks get smaller as fragment gets bigger -End of chart: get up to 1,000 bases in reliable sequence, peak quality degrades, Q value drops below acceptable, bp beyond should be ignored

Direct ELISA

-simplest format requiring antigen and enzyme conjugated antibody specific to antigen, involves detection of antigen using horseradish peroxidase conjugated antibody and luminol based enhanced chemiluminescence substrate -used to identify and quantify qualify antigen concentration in samples -advantages: less reagents needed/fewer steps making this the quickest ELISA and cross reactivity of secondary antibody is eliminated -disadvantaged: antigen immobilization is not specific resulting in potential high background interference and low flexibility- specific conjugated primary antibody is needed for each target protein -antigen is coated onto wells by passive adsorption -antibody conjugated with enzyme is added and incubated with antigen -substrate is added and color/signal develops

Identify heterozygous mutations in electropherograms

-software can compare 2 sequences to identify mutations Heterozygous mutations are observed as double peaks in the affected bp, must be confirmed by running the reverse sequence, can use reverse primer and forward primer if you have good ending crappy beginning and good beginning and crappy end- evens out Heterozygous deletions or insertions: affect all positions of sequence downstream of mutation, overlaid sequences will show minus bases if a deletion

Ion torrent Next-Gen sequencing platform

-uses ion-conductance sequencing -indexed libraries are amplified using primer immobilized on beads in aqueous oil emulsion -beads are placed on chip and captured bead are subjected to addition of nucleotides in predetermined order -if nucleotide is complementary to sequencing template, DNA pol will catalyze formation of phosphodiester bond -as this occurs, hydrogen ion is released and causes decrease in pH which is measured by detector

Automated Sanger sequencing. What is a call?

-way to make Sanger sequencing easier to read, uses fluorescent dyes instead of radioactive labels, 2 approaches: dye primer and dye terminator -label fragments synthesized during sequencing according to their terminal ddNTP, ddATP-green, ddCTP-blue, ddGTP-yellow/black, ddTTP-red, the color of the dye corresponds to the ddNTP that terminated the strand, fragments are then resolved by capillary electrophoresis -shorter fragments, closer to primer, will migrate faster than longer ones -detector will read fluorescence corresponding to each ddNTP as these pass through capillary -as fragments pass through capillary a laser excites the terminator dye and a signal is detected -software reads or calls these bases from smallest (fastest migrating fragments) that pass the detector, to the largest compiles these signals creating an electropherogram Applications of Sanger sequencing: microbial sequencing, plasmid sequencing, targeted DNA sequencing

Advantages and disadvantages of Maxam-Gilbert Sequencing

Advantages: simple way of determining which base pairs changed, showcases a lot of data, a lot of base pairs Disadvantages: do not want to do technique all the time, difficult to read and to write down, want to concentrate on other things

Identify AF in a paternity testing using RFLP or STR

DNA is inherited as one haploid chromosome complement from each parent each chromosome carries along its genes and the polymorphisms, thus each offspring inherits combination of parental polymorphisms in RFLP, band patterns represent combination of RFLPs inherited from each parent single locus will have several versions- alleles humans are diploid- 2 copies of every allele -homozygous- both these alleles are same -heterozygous- both alleles are different unique combination of RFLP in each individual can showcase parent's contribution of alleles to offspring from combination of alleles in subject and those of parents fragment sizes are combination of those from each parent Inclusion: alleged fathers are identified based on ability to provide the remaining alleles since in a paternity test the alleles (fragments) of offspring and mother are analyzed and remaining have to come from father

5 main steps of an EIA standard protocol

EIA: use specific antibodies to detect presence or absence of target molecules multiple formats: plate wells, strips, capillaries, all coated with capture antibody Step 1: Sample is added to the wells at corresponding dilution -if analyte is present in sample, this will be captured by immobilized antibody Step 2: Washing step Removes unbound molecules Step 3: Addition of secondary antibodies (detection Abs) -Secondary antibody covalently linked to marker, ex. alkaline phosphatase or horseradish peroxidase -Bind to another epitope of analyte in sample Step 4: Washing step -Removes unbound secondary antibodies Step 5: Development- substrate addition -Enzyme in secondary antibody will use the substrate to yield a signal; chemiluminescence, fluorescence, or color change -Intensity of signal can be measured in plate reader to calculate corresponding dilution

What is BLAST?

Finding the function of a gene from sequence: -used to identify biological role of the genes of interest- gene ontology -BLAST search: have a sequence that no genes have been identified, unknown sequence, and can find homology using another species using search

Next-Gen Sequencing applications

Gene expression analysis (RNA sequencing) Sequencing whole genomes: novel organisms, phylogenetics Discover novel RNA variants and splice sites

Components in HPLC

HPLC: High performance liquid chromatography, separates compounds dissolved in liquid and allows for qualitative and quantitative analysis of what components are there and how much -Migration rates of molecules differ with varying combinations of HPLC components and detectors 2 components: mixture of analyte (sample) in solvent- mobile phase column with chromatographic packing material- stationary phase Mobile phase: -organic solvents -injected with syringe through injection port in column -pumping molecules, force sample through column, then begin stationary phase Stationary phase: -size-exclusion columns -separation based on hydrophilicity -ion exchange- separation based on charge Reading: as mobile phase passes through column, a detector produced readout of signal peaks as sample components elute Detectors: light scattering, fluorescence, refractive index, UV absorption, and mass spectra

Explain heteroduplex analysis. Differentiate between hetero/homoduplexes and explain their migration pattern in a gel

Heteroduplexes: non identical ds DNA duplexes, migrate more slowly due to their sequence mismatches Homoduplexes: identical ds DNA duplexes mixing sample amplicons with reference amplicon, denaturing, and slowly renaturing (by increasing/decreasing temperature) -if sample contains mutant sequences fraction of renatures products will be heteroduplexes -if sample is identical to reference amplicon, homoduplexes will form structures are resolved in gel by electrophoresis and bans are analyzed

What is the purpose of preparing libraries for sequencing? What are adapters?

Library: collection of DNA fragments to be sequenced -in case of RNA, first need to convert to cDNA before preparing libraries, reverse transcriptase will allow you to synthesize DNA from RNA, since checking for gene expression only need mRNA- steps to select for it Step 1: purify and fragment mRNA Step 2: synthesize cDNA -cleaved mRNA fragments are copied into first strand cDNA using reverse transcriptase and random primers -second strand synthesis is followed using DNA pol to have dsDNA Step 3: ligate adapters- bar coding -multiple samples mixed and sequenced at once so sequencer needs to know which fragment corresponds to correct sample/specimen, cna identify each fragment by indexing/bar coding the samples -accomplished by adding adapters: short oligonucleotide sequences that are going to be added to each of the fragments in correspond sample -combination of adapters will be unique for each samples -adenylate 3'ends to ligate the adapter to the fragment Step 4: enrich DNA fragments -products are enriched with PCR and purified to create final cDNA -removes primers, enzymes, and other molecules from the reaction so that only cDNA is left -before sequencing, each sample must be normalized before pooling meaning that equal concentration of each of samples must be used for pooling which will ensure equal representation during sequencing

What are ddNTPs? How are these used in Sanger sequencing?

Modified dideoxynucleotides, they are nucleotides -added to the reaction, DNA elongation will stop when incorporating them, causing chain termination Sanger sequencing: -primer is covalently attached at 5' end to 32P labeled nucleotide or fluorescent dye labeled nucleotide -DNA replication to make full length copies of DNA template -1:1 mix of template and labeled primer is put into 4 reaction tubes, each with same ingredients for PCR reaction, mixture of all 4 dNTPS and one of 4 ddNTPS are added to each tube, each tube corresponds to each tube of nucleotide -add DNA pol, after 20 min terminate reaction by adding stop buffer -sets of synthesized fragments are resolved in gel- sequencing ladder How to write sequence from Sanger sequencing gel: -ladder is read bottom to top -smallest fragment represents first nucleotide attached to primer -band is formed at each interval when elongation stopped, corresponding ddNTP in tube

What are contigs?

Next-gen sequencing- mapping: -each read must be mapped to reference genome -as reads overlap, these form contigs: contiguous sequences

What is coverage?

Next-gen sequencing- raw data: -Coverage: covers all regions of the genome sequencing must have enough coverage meaning the reads cover the regions of the genome

What is a read?

Next-gen sequencing- raw data: -Read: data string of A, T, C, and G bases corresponding to the sample DNA or RNA, typical run can yield total of 350-500 mil reads each 125 bp in length

What are .fastq files? How are these different from .fasta files?

Next-gen sequencing: -raw data: uploaded to cloud storage, sample demultiplexed according to adapters, raw reads are obtained by .fastq format which includes quality (Q) score -FASTQ files: raw data with base call and quality (Q) score, used as sequence input for alignment in an analysis software, converted to FASTA files -FASTA file: contains list of genes that were sequenced along with their nucleotide sequence and organism name

What are the properties of the genetic code?

Redundancy: more than 1 codon can code for the same amino acid Wobble: base pairing is more flexible in the 3rd base of the codon -codons are located in the mRNA -anticodons are located in tRNA, these are complementary to the codons -negative aspect of genetic code is that it doesn't take much to change to another amino acid

What is transcriptomics?

Transcriptomics-using Next-Gen sequence for gene expression analysis Goal of project: assess gene expression in mice in response to vaccination over time Gene expression analysis-workflow: -treat animals and collect specimens -extract total RNA from spleen -cDNA synthesis of libraries -library normalization and Next-gen sequencing -analysis of differential gene expression -pathway mapping and gene network analysis -validation- qRT-PCR

What are GWAS?

Using next-gen seq for genome wide association studies (GWAS) Genome Wide Association Studies (GWAS): combines epidemiological and whole genome data from multiple individuals to identify set of genetic variants to see if any of these is associated with trait (such as a disease)- common disease/common variant

Explain sonication and nebulization in whole genome sequencing:

Whole genome sequencing: -for library prep, whole DNA must be cut into small pieces for library prep which is called fragmentation Sonication: shearing DNA with high frequency acoustic energy Nebulization: forcing DNA in suspension through small opening produce DNA fragments 100-1,000 bp that are used for library prep

What is bin? genotyping considerations

acceptable range of sizes and their distribution; all peaks or bands that fall within this window are considered identical

Identify STRs as markers of disease

as locations of many STRs in genome are known, these can be used to map genes, especially those genes associated with disease if linkage is close to the gene, STR may serve as marker for disease testing instead of testing for mutations in disease gene, marker allele (STR) is determined easier to look for linked STR allele than to screen large gene for point mutations presence of indicator STR allele serves as genetic marker for disease

How many loci are used in parentage testing?

at least 8 loci are used in parentage testing the more loci, the higher the probability of positive identification of the father

What are some of the limitations of MALDI-TOF for pathogen ID?

cross contamination high maintenance databases are proprietary tiny colonies may fail identification Advantages: reduced need for media and reagents, reduced expenses, faster turnaround time

RFLP typing

differences in the sizes and number of fragments generated by RE digestion of DNA change in nucleotide sequence, can alter restriction site making the RE unable to cleave the DNA at site or it can create new restriction sites create restriction map compare number and sizes of fragments expected based on restriction map polymorphisms are detected by observing fragment numbers and sizes different from those expected from map can be combined with Southern blot to add probes that identify specific sequences within RE sites

What is genetic concordance?

event where all alleles from 2 sources are the same- indicates inclusion of single individual as donor of both genotypes, 2 samples are considered different if at least 1 locus differs- exclusion

Silent mutations

lead to no change in polypeptide, results from redundancy in genetic genetic code

STRs

microsatellites, short tandem repeats short blocks of repeated sequences 1-10bp

VNTRs

microsatellites, variable number tandem repeats repeats of 10-50bp

What is the purpose of using a reference genome in mapping?

need good enough coverage (overlapping fragments) align with reference genome to have good sequence read of next-gen sequencing

Identify electropherograms with no DNA

no template shows as random noise, peaks very short which indicates no DNA

SINEs

polymorphic repetitive elements, repeated sequences that may be inverted, deleted, or duplicated form one individual to another short interspersed nucleotide element

LINEs

polymorphic repetitive elements, repeated sequences that may be inverted, deleted, or duplicated from one individual to another long interspersed nucleotide elements 6-8kbp

Calculate probability of paternity

probability of paternity = CPI x .50 (prior odds)/(CPI x .50) + (1-.05)

What is de novo assembly of genomes/transcriptomes?

researcher can search unknown sequence using blast

RFLPs

restriction fragment length polymorphisms, one or more nucleotide change that affects the size of the restriction enzyme products

SNPs

single nucleotide polymorphisms, most common type of genetic variation each SNP represents difference in single nucleotide in DNA sequence, occur once every 1,000 bp

Explain the procedure of MALDI-TOF for ID of microbes

uses direct colony testing, colony is picked from culture plate to a spot on MALDI TOF MS target plate matrix assists in desorption and ionization of microbial analytes through energy of laser as result of being shot by laser, microbial and matrix molecules are desorbed converting it to ionized state, ionized molecules accelerated based on mass to charge ratio into mass analyzer detector reads smaller analytes reaching detector first followed by larger analytes mass spectrum is generated representing number of ions of given mass impaction the ion detector ribosomal proteins are main contributors to generated mass spectrum specific proteins not identified mass spectra unique to individual organism types, with peaks specific to species and strains most closely related organisms are identified with value of level of confidence in identification

Nanopore

uses membrane diffusion through pore to detect changes in voltage according to nucleotides as they pass through transporter protein