final - intro to molecular diagnostics (cls 605)

DNA sequencing; amplification; hybridization techniques; etc

DNA sequencing (definition)

• Methods used to determine the order of nucleotides in a DNA molecular

• "gold standard" for mutation analysis

first generation DNA sequencing (general)

• In the 70's, two ways to sequence DNA were developed

• Maxam Gilbert sequencing

  • Uses a series of chemicals that cut DNA into fragments at certain bases

• Sanger sequencing

  • Chain termination method using dideoxynucleotide chemistry

  • more popular method, still heavily used today

basic steps of sanger sequencing

1. extract DNA

2. denature DNA

3. anneal primer to template DNA

4. synthesis and incorporation of deoxynucleotides and fluorescently labeled dideoxynucleotides (chain termination)

   • steps 2-4 repeated 30-40 times

5. detection and sizing of fragments

(sanger sequencing) DNA denaturing step

dsDNA is heated so that the strands of DNA separate

(sanger sequencing) annealing of primer to template step

• system cooled to annealing temp

• only a single primer is used—there is NOT a primer for the complementary strand

(sanger sequencing) synthesis of new DNA step

• The following things are added:

  1. DNA polymerase

  2. dCTP, dTTP, dGTP, dATP (regular DNA nucleotides)

  3. small amounts of ddCTP, ddTTP, ddGTP, ddATP

  4. DNA pol synthesizes complementary strand using the dNTPs but occasionally adds a ddNTP instead which causes chain termination (ddNTP has no 3’ OH but fluoresces when added)

(sanger sequencing) detection & sizing of fragments

• old method used radioactive tags instead of fluorescent tags which required 4 separate reaction tubes

• new method: after 30-40 cycles, products separate on a capillary and screened for fluorescence (see both pics)

(sanger sequencing) sequence alignment

way of arranging the sequences of DNA to identify regions of similarity that may be a consequence of functional, structural or evolutionary relationships between the sequences

• BLAST (basic local alignment search tool) compares an input sequence with sequence in a selected database

limitations of sanger sequencing

• can only sequence short pieces of DNA--it doesn't work well for sequences longer than 1000 base pairs

• quality is often not very good in the first 15-40 bases because that’s where the primer binds

• Sequence quality degrades after 700-900 bases

• Low throughput & limited read length

• Not accurate in GC-rich and repetitive regions

• Relatively fast and cheap for short sequences but inefficient and expensive for long sequences

next-generation (2nd) sequencing

• aka massively parallel sequencing

• Sequencing large numbers of templates carrying millions of bases simultaneously

• Several high-throughput approaches to DNA sequencing

  • Pyrosequencing

  • Reversible dye terminator

  • Ion conductance

(next gen sequencing methods) pyrosequencing

• Detects incorporation of nucleotides during DNA synthesis by monitoring light emitted from a chemiluminescent reaction

• Sequencing by synthesis

• Reaction mix: ssDNA template, primer, sulfurylase, luciferase, adenosine 5' phosphosulfate (APS), luciferin

• example: Roche 454

general steps of pyrosequencing (2nd gen)

1. Sample preparation—DNA extracted and fragmented

2. PCR amplification—DNA Is amplified, one strand is labeled

3. Sequencing reaction—light is generated when base is incorporated

4. Sequence analysis—light signals are analyzed to determine sequence

chemical principle behind pyrosequencing

• dNTPs added one at a time—if the nucleotide is complementary to template at next base, DNA polymerase extends primer

• Pyrophosphate (PPi) is released when phosphodiester bond forms

• PPi converted to ATP by sulfurylase

• Luciferase converts luciferin → oxyluciferin

• Light released

• If nucleotide was not incorporated, it gets degraded by apyrase

• Process is repeated

(next gen sequencing methods) reversible dye terminator

• Uses fluorescently labeled nucleotides with reversible blocking group

• Sequencing-by-synthesis

• General steps

  1. Sample prep

  2. Immobilization and amplification

  3. Sequencing

  4. Data analysis

(reversible dye terminator method) Illumina MiSeq

• 1 million to 25 million reads per run

• Run time: 4-56 hours

• Output: 540 mb-15 Gb

• Lots of short reads

(reversible dye terminator method) illumina library generation

• Fragmentation of DNA

• End repair of fragmented DNA

• Ligation of adapter sequences

• Optional library amplification

(reversible dye terminator method) adaptors & cluster generation

• adaptors: short synthetic DNA sequences added to the DNA fragments

  • Allow attachment of DNA to flow cell

  • Priming sites for amplification

  • Sample indexing (like a barcode)--can run multiple DNA at once

• cluster generation: clonal copies of forward and reverse strands in a cluster (500-2000 copies)

  • increases the signal

(reversible dye terminator method) illumina sequencing steps

DNA polymerase, connector primers and 4 dNTP w/ base-specific fluorescent markers added to reaction system—3'-OH of these dNTP are blocked, which ensures that only one base will be added at a time

1. Add bases

2. 1 base gets incorporated

3. Remove unincorporated bases

4. Detect signal

5. Unblock

6. Repeat

• high sensitivity camera captures fluorescent signal emitted by the incorporated nucleotide, identifying the base

(next gen sequencing methods) ion torrent sequencing

• Does not use measure light or fluorescence

• Uses semiconductor chips to detect when hydrogen atoms are released during DNA synthesis (pH changes)

steps of ion torrent sequencing (2nd gen)

• Beads with amplified DNA are loaded into wells on semiconductor

• One at a time nucleotides are flooded across the chip

• If that nucleotide gets incorporated, hydrogen ions are released

• Ion sensor detects change in pH and converts to electrical signal

(next gen sequencing) read depth

the number of times a specific nucleotide is sequenced

• 10x read depth means that each nucleotide was sequenced 10 times

  • Helps with alignment, accuracy and reliability of the results

  • Increases the confidence that the sequencing is working and that the bases added are accurate

3rd generation DNA sequencing

• main difference from 2nd gen: can take longer reads

• instruments/technologies: oxford nanopore & pacbio

(3rd gen sequencing) oxford nanpore

• Uses engineered proteins to physically ID bases

• Nanopore made from the protein alpha hemolysin

• Cyclodextrin is localized within the nanopore

• Cyclodextrin transiently binds with molecules as they pass through the nanopore

oxford nanpore steps

1. lipid bilayer created over a microwell containing a pair of electrodes on both sides

2. nanopores introduced into the bilayer, creating holes

   • lipid bilayer has a high electrical resistance so current flows only through the nanopore

3. DNA sample is introduced into the top layer

4. electrical field pulls charged particles through the pore; particles transiently bind to the cyclodextrin and produce a change in impedance proportional to the volume of the particle

5. bases are detected and identified by their characteristic impedance change

(3rd gen sequencing) pacbio general info

• 1 million to 25 million reads per run

• Run time: 12-30 hours

• Output: 120-480 Gb

• Long reads

steps of the pacbio (3rd gen)

• Involves a single stranded molecular of DNA, bound to a DNA polymerase enzyme

• The bound pair enter a sequencing chamber, called a flow cell

• Like in Sanger sequencing, the DNA polymerase adds complementary, fluorescently labelled bases to the DNA strand

• As each labelled base is added, the fluorescent color of the base is recorded before the fluorescent label is cut off, the next base in the DNA chain can then be added and recorded

target vs signal amplifcation

• Target amplification: increases the number of target molecules

• Signal amplification: increases the signal generated by a fixed amount of target

signal amplification methods (4)

1. Branched DNA (b-DNA)

2. Hybrid capture

3. Cleavage-based amplification (invader)

4. Cycling probes

(signal amplification methods) branched DNA (b-DNA)

1. Short oligo probe captures target nucleic acid onto solid surface

2. Extender probes bind to target

3. Multiple reporter probes bind to extender probes--reporter probes bound to alkaline phosphatase labeled nucleotides

4. Dioxetane (alk phos substrate) added, reaction produces light

branched DNA advantages over PCR + applications

• Advantages:

  • Fast

  • No need for thermocycler--can do at one temperature

  • Lower contamination risk--doesn't make a ton of copies that are now floating around

  • Less chance for nonspecific binding (more specific)

• Applications:

  • Hepatitis B, C testing

  • HIV testing

(signal amplification methods) hybrid capture

• Target DNA binds to ssRNA probe

• DNA;RNA hybrid recognized by antibodies

• Antibodies attached to solid surface and capture DNA;RNA hybrid

• Secondary antibodies attached to alkaline phosphatase added (sandwich assay)

• Substrate for alkaline phosphatase added--light produced

• Applications: HPV & genitourinary specimens, hepatitis viruses, CMV; also a hybridization technique

(signal amplification methods) cleavage-based/invader amplification

• Overlapping probes bind to target sequence (invader probe, signal probe)

• Cleavase cuts in overlapping region--cleaves signal probe

• FRET probe added (intact probe produces no signal) that has complementarity to the cut part of the signal probe

• Signal probe (now an invader probe) binds to FRET probe--due to folding of FRET probe this makes a three strand overlap

• Cleavase recognizes overlap and cleaves

• Signal gathered from FRET probe

(signal amplification methods) cycling probes

• probes made of DNA and RNA with a reporter at one end and a quencher at the other

• If complementary sequence is found, probe will anneal

• RNAse H cleaves RNA part of probe; releases reporter from quencher

• Applications:

  • Detect genes associated w antimicrobial resistance (vanA, mecA)

  • Detection of pathogens (herpes virus)

target based amplification methods

• usually known as isothermal amplification

  •  done at constant temp & does not involve temperature changes

• examples: transcription mediated amplification and nucleic acid sequence based amplification (TMA and NASBA), loop mediated isothermal amplification (LAMP)

(target/isothermal amplification) transcription mediated amplification

• Uses amplification through RNA instead of DNA

• Target molecule can be RNA or DNA

• Primers are designed to target a region of interest, but one primer includes the promoter sequence for T7 RNA polymerase at the 5' end

• If RNA Is the target: RNA → cDNA → RNA

• Benefits: RNA sensitivity prevents possible contamination

• Drawbacks: RNA is more sensitive to degradation

(target/isothermal amplification) loop mediated amplification (LAMP)

uses 4-6 primers recognizing 6-8 distinct regions of target DNA

• A strand-displacing DNA polymerase initiates synthesis and 2 specifically designed primers form "loop" structures to facilitate subsequent rounds of amplification thru extension on the loops and additional annealing of primers

• DNA products are very long (>20 kb) and formed from numerous repeats of the short (80-250 bp) target sequences

• can produce copies without need for denaturing the DNA

probe amplification methods

• Instead of increasing the amount of target molecule some methods increase the amount of probe

• Examples: Q-beta replicase, ligase chain reaction

(probe amplification) ligase chain reaction

• dsDNA denatured by heat → single-stranded target sequence

• temp lowered and 2 specific probes anneal to amplicon

• Another 2 probes bind to the amplicon's complementary sequence on the other strand

• When two probes anneal correctly to a target sequence, the 5' end of one probe is next to the 3' end of the other

• The probes can then be covalently joined by a heat-stable ligase to form a new target sequence--to which another pair of probes can subsequently bind

hybridization (definition)

annealing (base-specific hydrogen bonding) of a probe to a target nucleic acid to form a double stranded molecule

• probe: typically a single stranded DNA, RNA or PNA molecule, about 20-2000 bases in length

• target: ssDNA or RNA molecule

applications of hybridzation techniques

Because DNA probes with a known sequence will anneal to targets based on complementation, information can be gained about the sequence of the target DNA

• e.g. the presence or absence of mutations

• applications:

  • Southern blots (DNA is the target)

  • Northern blots (RNA Is the target)

  • In situ hybridization

  • Dot blots

  • Hybrid capture

  • Microarrays

western blot looks for _____ using _____?

looks for proteins using antibodies

northern blot looks for _____ using _____?

looks for RNA using RNA/DNA probe

southern blot looks for _____ using _____?

looks for DNA using RNA/DNA probe

(hybridization) probes

single stranded DNA, RNA, or PNA molecule about 20-2000 bases in length

• Complementary to target

• Usually labeled in some way

  • e.g. fluorescent molecule, ligand, isotope

• Common source of probes

  • Synthetic oligonucleotide

  • PCR product

  • Restriction endonuclease fragment

hybridization principle (pic)

southern blot

• Develop in 1975 by Edwin Southern

• Purpose: determines if there is a certain DNA sequence in a sample 

• General steps:

  1. Extract DNA

  2. Cut DNA with restriction enzymes

  3. Gel electrophoresis

  4. Blotting (transfer)

  5. Probe hybridization

  6. Detection

(southern blot) restriction enzyme cutting and resolution

• Enzymes used depends on gene of interest and application

• Digestion for extended amount of time to allow complete cutting

• Run gel electrophoresis with ethidium bromide

• Digested genomic DNA should look like a smear

• Problems:

  • Large aggregate of DNA at top = uncut

  • Smear mostly at bottom = degraded DNA

(southern blot) blotting/transfer step

• DNA transferred to solid surface so that you can then hybridize with the probe

• Possible substrates: nitrocellulose, nylon, modified cellulose, others

• Transfer methods: capillary (longest but most simple), electrophoretic, vacuum

(southern blot) pre-hybridization step

• After transfer, DNA may be permanently immobilized by baking or UV cross-linking

  • Prevents DNA from moving or washing away

• Pre-hybridization wash or blocking solution is added to prevent probe from non-specifically sticking to membrane

(southern blot) hybridization step

• Complementary to target gene

• Complementary sequences are not identical--they are antiparallel

• Example: if this is our target sequence (pic), what is probe sequence?

  • Answer: 5' ATCAGCGAGCTAC 3'

(southern blot) detecting probes (pic)

southern blot application—RFLP

• Differences in DNA sequences between/within individuals can result in variation in the position or cleavability of restriction endonuclease (RE) site

  • may be the result of mutations (and other changes) associated with specific genetic diseases/conditions

• These variations may be detected by examining the pattern or chromosomal RE fragments

(southern blot) RFLP applications

• Genetic disease/condition diagnosis

• A standard RFLP can be used in diseases:

  • Involving point mutations resulting in the loss (or gain) or restriction enzyme site

  • Deletion or insertion mutations involving at least 50 base pairs

  • Associated with altered DNA methylation

• Genetic identity testing

(RFLP applications) fragile X

• most common cause of inherited intellectual disability

  • Symptoms: physical and behavioral features and delays in speech and language development

• Caused by mutation of the FMR1 gene which codes for FMRP (fragile X messenger ribonucleoprotein)

  • FMRP is most actively synthesized in the brain

  • Females are less affected because they have two X chromosomes (and hence 2 copies of the FMR1 gene)

molecular diagnosis of fragile X

• Extensive repeat extension (>200) leads to methylation of C residues in CpG islands

• RFLP results from loss of cutting by methylation-sensitive restriction enzymes (e.g. Xhol)

• RFLP detected by hybridization with FMR probe

• Current diagnostic testing:

  • PCR as first line test

  • Southern blot (RFLP) as follow up

  • Why not use PCR only?

    • PCR doesn't give any information on methylation status and that is needed for diagnosis/severity

other blots/modifications of southern blot procedure

• Northern blot--used to detect RNA

• Western blots--uses antibodies to detect proteins

  • HIV confirmatory testing, lyme disease, variant Creutzfeldt-Jakob Disease (vCLD)

• dot blots

• microarrays

(hybridization) dot blots

• Simplified version of Southern or Western blotting

• Sample applied directly to membrane without electrophoresis first

• Presence and amount of target detected by use of labeled probes

  • What does a darker dot mean?

    • more of whatever you are detecting (semi-quantitative)

(hybridization assay) Digene HC2 HPV DNA Test

Screens for presence or absence of 13 types of high-risk HPV that are most associated with cervical cancer

• negative HPV test and normal Pap result provide confidence that a woman does not have, and is not likely to develop, high-grade cervical disease or cancer within the next 3 years (99.21% NPV)

(hybridization) microarrays

• A technology that can simultaneously detect thousands of specific DNA sequences in a short TAT

• two types: manufactured & spotted

• Applications:

  • Oncology: analyze gene expression in cancer cells

  • Infectious disease: detect the presence of nucleic acids from disease-causing agents

  • Mutation analysis: detect single nucleotide changes to large insertions or deletions

principle of microarrays

• Multiple probes are spotted to a solid surface (microscope slide or silicone chip)

• Hundreds to hundreds of thousands of different probes—may be representation of the entire genome, specific parts of a genome, infectious agents etc

• Fluorescently-labeled DNA from sample(s) is hybridized to the entire array of probes

• Array is scanned with a laser to detect hybridization

• The position of fluorescing spots identifies the nucleic acids present in the sample

microarray applications

• Infectious disease diagnosis

  • Probes for various pathogens

• Gene expression analysis

  • e.g. diagnose cancer type and treatment, identify genes expressed in response to drugs

• Sequence change identification

  • Identify SNPs associated with genetic disease

• Copy number imbalance identification

  • Array Comparative Genomic Hybridization (arrayCGH)

  • Identify chromosomal deletions and duplications

in situ hybridization

A technique to detect the presence or absence and location of specific DNA or RNA sequences within preserved chromosome preparations, fixed cells or tissue sections

• Can visualize chromosomal deletions, duplications, translocations, copy number variations (ploidy)

(in situ hybridzation) FISH for BCR/ABL

• Translocation between chr 9 and 22 (Philadelphia chromosome) assoc with CML

• Detected by hybridization with BCR and ABL probes

(hybridization) stringency

the tolerance for mismatch in base-pairing

• If a probe is complementary to a target sequence it will bind

• Stable base pairing requires about an 80% or greater match

high vs low stringency

• high: conditions where the probe must completely match the target sequence to bind

• low: conditions where the probe binds even if it doesn’t match the sequence very well

what happens in high stringency conditions?

detect less of target sequence but also less non-specific binding

what happens in low stringency conditions?

more detection of target, but more non-specific binding

(hybridization) manipulating stringency

• Temperature: increase temp increases stringency

• Na+ conc: increase conc = decrease stringency

• Formamide & urea: increases stringency

• "stringency" applies to both the hybridization steps and the subsequent washing steps

reasons for specimen rejection

• Incorrect labeling

• Not enough sample

• Wrong container

• Transportation issues

• Specimen issues (hemolysis)

• Specimens not received in time (esp for RNA)

considerations for infectious disease testing

• Type of sample depends on where the pathogen is found, replicates or sheds from

• Selection of sample type depends on organism, patient symptoms, and invasiveness of collection procedure

considerations for genetic disease testing

• Blood should be an acceptable specimen (i.e. white cells) to detect inherited mutations and to establish identity (paternity, forensics) as would be any cellular material (i.e. buccal smears, hair follicles, semen)

• Availability and/or ease of access may determine specimen used

considerations for cancer testing

• Malignant diseases usually require the specific tissue affection (ex: biopsy)

• However, mutations that make a person more susceptible to cancer (ex. BRCA1) can be detected with any cellular sample (blood)

(specimen requirements) southern blot

• Requires (a lot of) high quality DNA

• Fresh or quick frozen tissues

• If blood is sample type, need 5-10 mLs

(specimen requirements) PCR

• Can be done on degraded DNA

• Fixed/embedded tissues generally ok

• Freezing usually acceptable

(specimen requirements) FISH/ISH

• Typically done on intact preserved tissues

• Cells don't need to be living

• No freezing

(specimen requirements) karyotyping

• Need living cells

• Whole blood and bone marrow, need anticoagulant

• No freezing

(specimen requirements) next gen sequencing

• Usually 3-5 mLs of blood

• Freezing samples ok

if 100% of the DNA is recovered, how much DNA could be extracted from a 1 mL sample containing 5,000 WBCs/ul?

• (5000 WBCs/ 1 uL) x (1000 uL/1 mL) = 5,000,000 cells

• 5,000,000 cells x (1 ug DNA/151,000 cells) = 33.1 ug DNA

You are extracting DNA for a genomic analysis from a whole blood specimen containing 7,600 WBC/uL. You need 10ug of DNA for your procedure. Assuming you recover 50% of the DNA from the specimen, what is the minimum volume of blood you need for the analysis?

• 10 ug DNA x (151,000 cells/1 ug DNA) = 1,510,000 cells

• 1,510,000 cells x (1 uL/7600 cells) = 198.7 uL

• 198.7/0.5 = 397.4 uL

• 397.4 = 0.4 mL

(specimen collection; anticoagulants) EDTA

• preferred anticoagulant for PCR

  • EDTA chelates Mg2+, which inhibits nucleases

  • Not for karyotyping or cell culture

  • Can be stored at 4 deg C for 72 hours without DNA degradation, need to freeze for longer time periods

(specimen collection; anticoagulants) sodium heparin

• NOT FOR PCR

• Used for in situ hybridization and karyotyping

(specimen collection; anticoagulants) ACD

• Suitable for PCR but will create a dilution that needs to be accounted for

• Not acceptable for karyotyping

(sample types) tissue

• Total quantity of importance to provide adequate, extractable DNA

  • tissue quantity should be equal to the size of a pencil eraser ( 3mm x 3mm x 3mm)

• In PBS for collection (phosphate buffered saline)

• For transport, PBS drained and either dry ice or 70% ETOH used at RT

• Storage should be at 4 deg C

• Tissues must be received in viable and sterile condition if cell culture is needed (e.g. karyotype)

(sample types) swabs

• Swabs for PCR should be synthetic (no cotton or wood)

• Flocked swabs enhance sample collection

• Mucus should be removed (can be inhibitory)

(sample types) sputum

• Can be used for infectious disease testing

• Not ideal for other testing due to presence of normal flora and mucus

PCR inhibitors present in samples

• Blood—hemoglobin, IgG, lactoferrin

• Feces—bile salts

• Urine—urea

• Sputum—mucus

(transport/storage) RNA

• overnight shipping

• avoid changes in temperature while shipping

(transport/storage) whole blood/serum/plasma & bone marrow

• Whole blood = transport w/in 24 hours, store at RT for 24 hrs, refrigerate up to 72 hours

  • freezing generally not recommended

• Serum/plasma = usually for infectious disease testing

  • if testing for RNA viruses, spin and freeze immediately, ship on dry ice

• Bone marrow = handle like whole blood generally; refrigerate for up to 48 hours

  • freezing generally not recommended

(transport/storage) tissues & swabs

• Tissues = freeze at -20 deg C, transport on dry ice

• Swabs = generally transport in sterile saline or UTM (universal transport media), typically refrigerate for up to 72 hours after collection

  • freeze if specimen can't be processed within that time

storage of extracted nucleic acids

• DNA = can store in the refrigerator for up to a week, good for months or years  when stored at -20 or -70 deg

• RNA = store in refrigerator if using within a few days, store at -20 for use within a few months, store at -80 in liquid nitrogen for longer storage

principle of the biofire film array

• nested multiplex PCR

• creates melting curve to analyze results

• assays: respiratory, pneumonia, blood culture, GI, meningitis/encephalitis panels

principle of diasorin liaison instrument

• qualitative, quantitative, and multiplex PCR

  • no DNA extraction required

• assays: COVID-19, Flu A/B, HSV 1 & 2, Bordetella spp., Group A Strep

principle of the hologic panther

• proprietary real-time transcription mediated amplification to detect and quantify target sequences

• assays: women’s health & infectious disease panels (HIV, HCV, covid etc)

principle of the genexpert instrument

• catridge-based nested real-time PCR (qual/quantitative)

• numerous assays:

  • respiratory 

  • infectious disease; TB

  • virology and sexual health 

  • oncology and human genetics