Methods_02

1. 5’-ends of dsDNA are radioactively labeled by polynucleotide kinase and [g-32P]-ATP


2. dsDNA is denatured in the presence of DMSO and heat, and ssDNA is purified


3. Labeled ssDNA is split into 4 reactions, chemically treated and cleaved after specific bases

4. Obtained fragments are separated on a polyacrilamide gel and autoradiographed

• G
• A+G
• C+T
• C

Use deduction to compare the G results to A+G and the C results to the C+T results to decide which nucleotide it was

A dideoxynucleotide (for example, dideoxythymidine triphosphate, ddTTP) lacks 3’ hydroxyl group, 3′-OH

Lack of 3′-OH prevents the next nucleotide from attaching => DNA synthesis terminates

The newly synthesized DNA is separated by size in an electrophoretic field (either in a polyacrylamide gel or in a capillary)

The fluorescent dyes on the terminator nucleotides are activated by a scanning laser, tracked, and read by a photometer.

The original Sanger method used radioactively-labeled (and not fluorescently labeled dideoxy terminators

Use PCR to incorporate restriction sites



PCR amplify the genomic DNA fragment using extended primers that match the DNA of interest in their 3’ ends (~20nt) and harbor restriction sites of interest (e.g., EcoRI and XbaI) in their 5’ ends (~10nt)!

massively parallel sequencing

• Roche 454 pyrosequencing
• Illumina (Solexa) sequencing by synthesis
• Life technologies SOLiD sequencing by ligation
• Life technologies Ion Torrent semiconductor

Helicos Biosciences

Pacific Biosciences Single Molecule Real Time (SMRT)

Oxford Nanopore

Each probe is an octamer made of 2 probe-specific bases and six degenerate bases (nnnzzz) and a fluorescent label on the 5’ end

The three 5’ zzz nucleotides and fluorescent label are cleaved off with silver nitrate

The extension product is removed, and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles

Each primer enables reading of two out of every five nucleotides

(to read the other three nucleotides, the entire process is prefored again with a primer shifted with respect to the primer N by one nucleotide (N-1))

~20 nt in length, and Tm of 60-70C

Alkaline lysis depends on a unique property of plasmid DNA. It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.

LacZ acts as a reporter gene. Within the gene is a multiple cloning site. When the plasmid sucessfully takes in your gene of interest, the gene is inturrurpted and the colony remains white on the X-gal substrate.

• If the gene was not taken into the plasmid, the reporter gene will turn the colony blue
• 

E.coli that has been made to accept extraneous DNA either through chemical or electro-competent methods

Cells prepared by resuspending in CaCl2 to permeabilized E.coli cell membranes

Then cells are subjected to heat shock

used to make E.coli cells uptake plasmid DNA

prepared by washing the cells with water to remove salts and subjecting the cells to a short electric pulse

used to make E.coli cells uptake plasmid DNA

• For high-copy-number plasmids, random partitioning occurs
• For low-copy-number plasmids, replication is coordinated with chromosone replication
• 

• PCR
• Sanger squencing
• Restriction Digest

a dye commonly used to visualize DNA on a gel

EtBr-bound DNA glows under UV light, enabling visualization on a gel

• PCR rxn set up with forward vector primer and reverse insert-specific primer
• Primers will only amplify if the insert is present and in the right orientation
• 

• Microarray
• NanoString
• qPCR
• Optical mapping
• 

DNA microarrays have been used for genetic research since the early 1980s. In DNA microarrays, single-stranded DNA (ssDNA) probes are immobilized on a substrate in a discrete location with spots as small as 50 μm. Target DNA is labelled with a fluorophore and hybridized to the array. The intensity of the signal is used to determine the number of bound molecules.

Microarrays are used in many applications. Single-nucleotide polymorphism (SNP) arrays identify common polymorphisms associated with disease and phenotypes, including cardiovascular disease, cancer, pathogens, ethnicity and genome-wide association study (GWAS) analysis. Additionally, lower-resolution arrays are used to identify structural variation, copy number variants (CNVs) and DNA–protein interactions. Expression arrays measure expression levels by measuring the amount of gene-specific cDNA.

Microarrays remain widely used in genomic research. They are used to identify SNPs at costs far below NGS routines. This is also true for expression studies, in which arrays inexpensively measure expression levels of thousands of genes. Variations in hybridization and normalization are problematic, leading some people to recommend RNA sequencing (RNA-seq) over gene expression microarrays.

• Similar to microarrays, the nCounter Analysis System from NanoString relies on target–probe hybridization.
• Probes target a gene of interest; one probe is bound to a fluorophore 'barcode' and the other anchors the target for imaging. The number and type of each barcode is counted.
• NanoString is unique in that the probes are labelled molecules that are bound together in a discrete order, which can be changed to create hundreds of different labels.

nCounter applications are similar to those of microarrays and quantitative PCR (qPCR; see below), including gene expression analysis, CNV and SNP detection, and fusion gene detection. This approach provides exceptionally high resolution, less than one copy per cell, far below microarrays and approaching TaqMan in sensitivity. Unlike most NGS applications, neither template enrichment nor reverse transcription are required. Around 800 targets can be read at a time, far below either microarrays or NGS.

qPCR

• Real-time qPCR utilizes the PCR reaction to detect targets of interest.
• Gene-specific primers are used and the target is detected either by the
• incorporation of a double-stranded DNA (dsDNA)-specific dye or by the
• release of a TaqMan FRET (fluorescence resonance energy transfer) probe through polymerase 5′−3′ exonuclease activity.

Developed in the early 1990s, qPCR is widely used in both clinical and research settings for genotyping, gene expression analysis, CNV assays and pathogen detection. qPCR is extremely rapid and robust, which is beneficial for point-of-care applications. Its high sensitivity and specificity make it the gold standard for clinical gene detection with several US Food and Drug Administration (FDA)-approved tests. The number of simultaneous targets that can be detected is in the hundreds rather than the thousands for microarrays and NGS. This method also requires primers and/or probes designed for specific targets.

• Optical mapping combines long-read technology with low-resolution sequencing.
• Originally a method for ordering restriction enzyme sites through digestion and size separation, this technology now uses fluorescent markers to tag particular sequences within DNA fragments that are up to ~1 Mb long.
• The results are imaged and aligned to each other, and/or a reference, to map the locations of the probes relative to each other.

A central application of this technology is the generation of genome maps that are used in de novo assembly and gap filling. This technology can be used to detect structural variations that are up to tens of kilobases in length. Haplotype blocks that are several hundred kilobases in size can also be resolved.

Optical mapping can either be an alternative to NGS or a complementary approach. As an alternative, it provides a low-cost option for understanding structural and copy number variation, but it does not provide base-level resolution. As a complementary technology, optical mapping improves de novo genome assemblies by providing a long-range scaffold on which to align short-read data.

A flowcell contains 512 channels that thread 512 DNA molecules at once

~8kb but the error rate is high (8% for 2D reads, 20% or 1D reads)

• A single strand of DNA is threaded through a tiny protein pore in a synthetic membrane
• An electric current flows through the pore
• Different DNA bases disrupt the current in different ways
• The machine measures the current and interprets the sequence (aka “squiggles”)
• Drawbacks: higher error rates and lower throughput
• Pluses: portability and ease of use; can also sequence RNA

The theory behind nanopore sequencing is that when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it, an electric current due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases), strands of DNA or other molecules pass through or near the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.

85% vs 99%

Ligation of hairpin adapters to dsDNA fragments

(PacBio DNA prep involves ligation of hairpin adapters to dsDNA fragments essentially producing a SMRTbell, a circular template that is read by DNA polymerase multiple times, improving the accuracy of consensus sequence reads)

• A single DNA polymerase enzyme is affixed at the bottom of a zero-mode waveguide (ZMW) well with a single molecule of DNA as a template.
• The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase.
• Each of the four DNA bases is attached to one of four different fluorescent dyes on their terminal phosphate group.
• When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable.
• A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
• 

10-25kb on average, up to 40kb

Helicos Biosciences (defunct)

Pacific Biosciences Single Molecule Real Time (Pac Bio SMRT)

Oxford Nanopore

• This technology differs from other sequencing technologies in that no modified nucleotides or optics are used.
• Ion semiconductor sequencing may also be referred to as Ion Torrent sequencing, pH-mediated sequencing, silicon sequencing, or semiconductor sequencing.

• This is also a sequencing by synthesis approach (like Illumina and 454), but no modified nucleotides or optical detectors are used.
• The wells with template-attached beads are flooded with dNTPs, one kind at a time.
• When the nucleotide is incorporated into the growing complementary strand, a hydrogen ion is released -> a sensor reads this pH change as an electric signal.
• If homopolymer repeats are present in the template, multiple dNTP molecules get incorporated in a single cycle -> a larger number of protons is released, and a proportionally higher electronic signal is detected.

• This is a sequencing-by-synthesis approach that utilizes a DNA polymerization reaction to detect protons released during base incorporation
• 

MiSeq, NextSeq2000, NovaSeq6000

Library preparation and sequencing services

Illumina

1. 5’-ends of dsDNA are radioactively labeled by polynucleotide kinase and [g-32P]-ATP


2. dsDNA is denatured in the presence of DMSO and heat, and ssDNA is purified*


3. Labeled ssDNA is split into 4 reactions, chemically treated and cleaved after specific bases


4. Obtained fragments are separated on a polyacrilamide gel and autoradiographed**