Lab techniques

Detects all unbalanced chromosomes abnormalities, and copy number variants. It does not detect balanced abnormalities because balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number, which is what is detected by CGH technologies. Uses include, aneuploidy, duplication and deletion syndromes and tumour CNV analysis. 

Types of arrays : BAC (bacterial artificial chromosome) clones, Oligonucleotide clones, Genome wide clones, chromosome specific arrays, customised arrays. 

- Test and reference genomic DNA are differentially labeled with two different fluorophores. Cy3 green - test, Cy5 red - reference. Unlabelled COT-DNA is used to block normal repetetive sequences and the DNA is denatured. The test and reference DNA are hybridized competitively onto nucleic acid targets on the microarray plate. The microarrays are then washed and unhybridised DNA is washed away. Digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. The resulting ratio of the fluorescence intensities is proportional to the ratio of the copy numbers of DNA sequences in the test and reference genomes. If there is an altered Cy3:Cy5 ratio this indicates a loss or a gain of the patient DNA at that specific genomic region.

The main disadvantage of array CGH is its inability to detect aberrations that do not result in copy number changes and is limited in its ability to detect mosaicism. The level of mosaicism that can be detected is dependent on the sensitivity and spatial resolution of the clones. At present, rearrangements present in approximately 50% of the cells is the detection limit. For the detection of such abnormalities, other techniques, such as SKY (Spectral karyotyping) or FISH have to still be used.

 Another disadvantage is the lack of commercial availability of the arrays. Until now, BAC and cDNA clones can be obtained from, for example, the Sanger Center, BACPAC Resources, Invitrogen and Research Genetics.

However, array preparation still needs to be performed by the investigators themselves. Inconsistencies in visualization and imaging software as well as interpretation parameters also make it difficult for replications and comparisons to be made by different laboratory teams.

May still be unable to detect small pathogenic imbalances. 

'Chain Termination Method' 

-requires a single-stranded DNA template, a DNA primer, a DNA polymerase, 4 different dNTPs, and fluroesently labelled modified di-deoxynucleotidetriphosphates (ddNTPs). The ddNTPs are chain terminating so lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA. 

The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while the other added nucleotides are ordinary ones. The DNA binds complementary to the primer, and elongation occurs until chain termination by the ddNTP meaning you get many fragments of differing lengths. 

Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis or are analysed by laser detection of fluorochromes for each lane - A T G and C. 

Common challenges of DNA sequencing with the Sanger method include poor quality in the first 15-40 bases of the sequence due to primer binding and deteriorating quality of sequencing traces after 700-900 bases. Additionally formation of DNA secondary structures which alter sequencing fidelity can occur. 

•Up to 700bp per read

•Whole gene testing (smaller genes only – some large exons may need several sets of primers)  

•Used for looking for specific variant (eg. familial studies)

The larger the fragment, the less the separation and resolution so normally 700bp is the limit. 

Longer sequences are subdivided into smaller fragments that can be sequenced separately, and subsequently they are re-assembled to give the overall sequence.

In shotgun sequencing, DNA is degraded non specifically by sonification or DNAase digestion into numerous small segments, degradation is controlled so the reads are around 2000 bp. These fragments are cloned into a sequencing vector which is sequenced to 6 fold. 500 bp from each end of each fragment are decoded generating millions of sequences. Sequencing both ends of each insert is critical for the assembling the entire chromosome.Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence

- can sequence larger read lengths than sanger sequencing

 It relies on the detection of pyrophosphate release on nucleotide incorporation

The desired DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that only one out of four of the possible A/T/C/G nucleotides are added and available at a time so that only one letter can be incorporated on the single stranded template (which is the sequence to be determined).DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) which results in an enzyme reaction where light is emitted.The intensity of the light determines if there are more than one of these "letters" in a row. The previous nucleotide letter (one out of four possible dNTP) is degraded before the next nucleotide letter is added for synthesis: allowing for the possible revealing of the next nucleotide(s) via the resulting intensity of light (if the nucleotide added was the next complementary letter in the sequence). This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined.

Read ~400 bases per sequence run (smaller than Sanger)

Solexa HiSeq 2500. Generates **DNA fragments ~200 - 300 bp. Tagged with an ‘A’ and ‘B’adapter. Amplified on Flow Cell by Bridge PCR to form clusters. 

Prepare genomic DNA sample - randomly fragment genomic DNA and ligate adapters to both ends of the fragments.  Attach DNA to surface - bind **fragments randomly to the inside surface of the flow cell channels.  Bridge amplification - add unlabeled nucleotides and polymerase to initiate solid phase bridge amplification.  Fragments become double stranded - polymerase incorportates nucleotides to build dsbridges on the solid phase substrate.  Denature the ds molecules - leaves ** templates anchored to the substrate.  Complete amplification - several million dense clusters of ds DNA are generated in each channel of the flow cell. Determine the first base - the first seuencing cycle begins by adding four labeled reversible terminators, primers and DNA polymerase.  Image first base - after laser excitation the emitted flouresence from each cluster is captured and the first base is identified. This is repeated over many cycles to determine the sequences of bases in a fragment one base at a time. 

Can sequence 8 human genomes in 10 days. 

Uses detection based on PH change. Choice of 3 chips which sequence 10Mb, 100Mb or 1Gb. 

A microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide triphosphate (dNTP). If the introduced dNTP is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers an ISFET ion sensor, which indicates that a reaction has occurred.

Adding an deoxyribonucleoside triphosphate (dNTP) into a growing DNA strand involves the formation of a covalent bond and the release of pyrophosphate and a positively charged hydrogen ion. A dNTP will only be incorporated if it is complementary to the leading unpaired template nucleotide. If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The hydrogen ion that is released in the reaction changes the pH of the solution, which is detected by an ISFET. The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.

The major benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.This has been enabled by the avoidance of modified nucleotides and optical measurements.

Because the system records natural polymerase-mediated nucleotide incorporation events, sequencing can occur in real-time. 

In nanopore sequencing, the nanopore is embedded in a membrane to create a passage for the ionic solution. When a constant voltage is added over each side of the membrane, an ionic current through the passage is detected and drive ssDNA or ssRNA to pass through the pore. The polynucleotides are bounded with polymerase or helicase enzyme to control the movement speed of nucleotide. The narrowest region of nanopore is most sensitive. The nucleobase's mass and its associated electrical field will change the ionic conductivity of the sensitive region, resulting in a current level variation. The ionic current levels reveal the sequence of strand and can be measured by a sensitive ammeter.

Advantages - no PCR, no dyes, no hybridisation. 

No labs have the technology yet. 

The zero-mode waveguide (ZMW) is a nanophotonic confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate

The DNA sequencing is done on a chip that contains many ZMWs. Inside each ZMW, a single active DNA polymerase with a single molecule of single stranded DNA template is immobilized to the bottom through which light can penetrate and create a visualization chamber that allows monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a phospho-linked nucleotide incorporated by the DNA polymerase is detected as the DNA synthesis proceeds which results in the DNA sequencing in real time. Each of the four DNA bases is attached to one of four different fluorescent dyes. 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.

Used for epigenetics, de novo sequencing, transcriptomics

SSCP is able to detect mutations of the following nature:

·       Single base substitutions, Small insertions/deletions, Micro-inversions. It is therefore a widely used technique as these mutations contribute to a large number of genetic disorders. 

1.The DNA sequence of interest is amplified using the Polymerase Chain Reaction (PCR). This is often accompanied by the labelling of DNA with radioisotopes of dNTPs (32P, 33P). 2.The amplicons are then denatured to obtain single strand DNA molecules. This can be done by heating at 95C, or by use of chemicals such as NaOH or formamide. 3.Denaturation is immediately followed by rapid cooling of the ssDNA in ice water. This prevents the individuals strands from re-annealing with their complements. It also allows the ssDNA to fold into unique conformations, upon which this technique is based. 4.The mobility shifts/differences of the ssDNA can be detected by electrophoresis, either gel or capillary. It is essential that the gel electrophoresis is under non-denaturing conditions, i.e. native PAGE. Denaturing conditions will disrupt the second structure of the molecules, making it unable to distinguish between mutant and wild type.1 If the PCR products were not previously labelled, silver staining or use of fluorescent dyes (such as EtBr) can be used to observe the banding patterns. Silver staining allows analysis under ambient light, unlike fluorescent dyes (such as Ethidium Bromide) which can only be viewed under UV light. EtBr also has less efficiency binding to ssDNA and is hence not preferred.

     It is only capable of detecting the presence of the mutation, it is unable to provide us with information regarding the type/characteristic of the mutation. In addition to this, it is a highly sensitive technique, therefore conditions of the experimental procedure must be closely monitored to ensure success, as well as reproducibility. 

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a multiplex methodology that incorporates the standard quantitative real-time PCR and the reverse transcriptase PCR.  It is the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.

In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR.Therefore, when the cDNA is transcribed, DNA primers and probes hybridize and anneal to their allocated regions opposite the cDNA, ready for transcription of the new DNA strand. The DNA polymerase (ideally used Taq polymerase) binds to the primers and precedes the extension of the strand. Simultaneously, the probes that anneal to the cDNA strand have a linear structure, with a reporter end domain and a quencher domain at the opposite end (a commonly used probe is TaqMan). These probes are specific to DNA strands and help quantify the amplification of DNA. The reporter end is a florescent marker for the positive PCR; however, the quencher domain restricts its activity when bound together. Therefore, as the DNA polymerase begins to transcribe the cDNA and runs along the RNA strand, it cleaves the probe upstream, releasing the reporter end, which then begins to fluoresce, highlighting the transcription of the DNA strand (Figure 1). This fluorescence is measured in real time to quantify the new DNA strand. 

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. 

The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. 

TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers.  As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Pyrosequencing bisulphite modified DNA is a quantitative methodology commonly used to study methylation within a sequence of up to 100-bp in length.

Bisulphite treatment of DNA renders unmethylated cytosine into uracil but leaves unmethylated cytosine intact. The pyrosequencing reaction requires a biotin label that isolates a single strand template of this bisulphite-modified DNA. Single nucleotides (dNTPs) are then added in a predetermined order in each cycle of the pyrosequencing process resulting in a proportional emission of light (pyro-) at individual CpG positions in a strand-dependent manner that is then expressed as a peak-graph. The results are displayed as an average for each CpG position assayed across all PCR products. As a result of this, bisulphite pyrosequencing allows for the identification of heterogeneous DNA methylation patterns at the expense of single allele resolution. Bisulphite pyrosequencing is generally most suited for the analysis of short DNA sequences (e.g. for formalin-fixed paraffin-embedded samples). However, serial bisulphite pyrosequencing can be used to sequence longer PCR products.   

ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest.

I. CHROMATIN IMMUNOPRECIPITATION
1. DNA-Protein cross links are formed using Formaldehyde.2. DNA, which is sensitive to sonication, is sheared to obtain fragments ~ 500- b.p. 3. A suitable antibody specific to the protein is added to the solution. 4. The antibody bound protein-DNA complexes are captured using magnetic/Sephadex beads. 5. Reverse the cross linking via Proteinase K, DNAse, etc.

II. LIBRARY PREPARATION6. Quality control of the ChIP DNA depending on the protein being studied. 7. PCR 8. Quality check of the PCR products

III. NGS SEQUENCING
IV. DATA ANALYSIS >Illumina’s Genome Analyzer

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive defence mechanism protecting many bacteria and archaea from invading plasmids and phages. CRISPR loci contain a combination of CRISPR-associated (Cas) nuclease genes as well as non-coding RNA sequences that determine the specificity of the Cas nuclease-mediated cleavage of foreign DNA. 

In type II CRISPR-Cas systems, two non-coding RNAs, crRNA and tracrRNA are transcribed in the first step. The tracrRNA then hybridises to crRNA and mediates its processing into a mature crRNA. The mature crRNA:tracrRNA complex (or dual guide RNA, gRNA) directs the Cas9 nuclease to the target foreign DNA by complementary base pairing of its spacer regions and protospacer regions on the target DNA. The Protospacer Associated Motif (PAM) lies on the 3’ end of the protospacer sequence and is essential for target DNA recognition. Cas9 cleaves both DNA strands in the protospacer region.

The simplicity of the CRISPR-Cas9 system bears an immense potential for the design of genome engineering experiments. Only three components are required to efficiently cut dsDNA – crRNA, tracrRNA and Cas9.

A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.

Mass spectrometry is an analytical technique that measures the molecular mass of a sample. It identifies the amount and type of molecules in a sample by applying an electric and magnetic field to charged ions in order to measure their mass-to-charge ratio and abundance in the sample.

A mass spectrometer is made up of 3 key components: The ionization source, the mass analyzer and the detector. The sample is first introduced into the ionization source where it is ionized and subsequently picks up a charge. The ions are then accelerated to the mass analyzer where they are sorted and separated according to their mass-to-charge ratios. Finally, they are detected and analyzed by the detector, which converts the ion signals into a mass spectra. 

Applications of mass spectrometry include getting accurate molecular weight measurements, reaction monitoring, amino acid sequencing, oligonucleotide sequencing, and detection of protein structure. 

The Infinium Methylation Assay detects cytosine methylation at CpG sites based on highly multiplexed genotyping of bisulfite-converted genomic DNA (gDNA).

Upon treatment with sodium bisulfite, unmethylated cytosine bases are converted to uracil, while methylated cytosine bases remain unchanged. The Infinium I use fluorescence from two different probes, unmethylated (converted) and methylated (unconverted), to assess the level of methylation of a target CpG.

If a target CpG was methylated in the sample, the DNA fragment will remain unconverted after bisulfite treatment and will therefore bind to the complementary ‘methylated’ probe, which terminates at the 3’ end with a cytosine. If the target CpG was unmethylated, however, binding will occur to the complementary ‘unmethylated’ probe, which terminates at the 3’ end with a thymine. Binding at either probe is followed by single base extension that results in the addition of a fluorescently labeled nucleotide. It is assumed that the methylation status of CpGs underlying the 50 bp probe body is correlated to that of the target CpG such that CpGs in the probe body of an unmethylated (converted) probe are also converted, while CpGs in the body of a methylated (unconverted) probe will remain unconverted.

 Methylation state is detected by single base extension at the position of the ‘C’ of the target CpG, which always results in the addition of a labeled ‘G’ or ‘A’ nucleotide, complementary to either the ‘methylated’ C or ‘unmethylated’ T, respectively. The Illumina HiScan or iScan System scans the BeadChip, using a laser to excite the fluorophore of the single-base extension product on the beads. The scanner records high- resolution images of the light emitted from the fluorophores.

MLPA is the gold standard for DNA copy number quantification. It can be used to quantify different DNA sequences from only 20ng of DNA.

MLPA is a special form of multiplex PCR in which a MLPA probe is amplified instead of the DNA sample. To perform MLPA only a thermocycler and a sequence type electrophoresis system is required. Each MLPA probe consists of two oligonucleotides that can be ligated to each other if the probe target sequence is present in the sample (i.e. when they’re hybridised to the target sequence). One of the oligonucleotides has a common sequence used for PCR amplification at the 5’ end and a target-specific sequence at the 3’ end. The other probe has a target-specific sequence at the 5’ end which is immediately adjacent to the first oligonucleotide, a common sequence used for PCR amplification at the 3’ end and a stuffer sequence (to obtain sufficient probe length). One PCR primer is fluorescently labelled, enabling the amplification products to be visualised during fragment separation.

Applications: Dosage analysis of cancer predisposition genes, identification of deletions and duplications in BRCA1 and BRCA2 genes. Prenatal diagnosis of deletion syndromes, Duchenne Muscular Dystrophy where 65% of mutations are deletions. Detection of microdeletion syndromes egWilliams syndrome, Prader-Willi-Angelman and DiGeorge.

MS-MLPA – detection of methylation changes, Imprinting, Epigenetics 

RNA interference (RNAi) RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.

Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression.

Post transcriptional gene silencing. 

RainDance Technologies offer a digital micro droplet-based PCR where a machine, called the RainDrop source machine, generates 1,000,000s of picolitre sized droplets in each sample. It does this by oil emulsion which enables the droplets to stay separate. Each droplet effectively acts as a PCR tube in that it contains a PCR reaction. The RainDrop sense machine is then used to quantify the amount of PCR product(s) present it does this by detecting fluoresnce in the samples. This can detect up to 10 targets per sample by using two different colours of fluoresence and having different intensities of fluoresence correspond to different targets.

Applications where dPCR has been demonstrated or may be well suited include: rare allele detection in heterogeneous tumors or other genetic-based diseases [11]; liquid biopsies of solid tumor burden using peripheral body fluids [12]; non-invasive prenatal diagnostics [13]; viral load detection [14]; gene expression; copy number variation in heterogeneous samples [9]; assays with limited sample material, such as single cell gene expression and FFPE samples; DNA quality control tests before sequencing [15,16]; and validation of low frequency mutations identified by sequencing.

SNP arrays produced by Affymetrix and Ilumina work by hybridisation, where fragmented, single-stranded target DNA sequences bind to probes that are immobilized on the array by complementary base pairing. The intensity of the signal associated with the hybridisation of the target sequence and the probe is measured by specialized equipments and analyzed using computational algorithms.

SNP stands for single nucleotide polymorphism, which is a variation in a single nucleotide that occurs at a specific position in the genome, and is present at a minor allele frequency (MAF) of more than 1% in the population. SNPs can be found in coding regions, non-coding regions or intergenic regions in the genome. SNPs underlie our susceptibility to diseases, the severity of illness and the way our body responds to treatment.  

 Ancestry determination from multiple genotypes. Important in disease association studies, where population stratification may lead to false apparent association when the affected and unaffected groups have different ancestry. • Pooled DNA and allele-specific expression. Since SNP arrays can generate many genotypes quickly, SNP allele frequencies can be measured by using array signals (from a large group of individuals) as a continuous variable instead of SNP genotypes, a categorical variable.  • Genome wide association studies. To collect data from a large number of people and find variants that are more prevalent in individuals with disease than those who are free of disease. • Detecting somatic cell changes in cancer cells. For example, loss of heterozygosity and copy number variations. 

Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence.

In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays.

Reporter gene assays are used to investigate the genetic regulatory elements that control the expression of genes of interest. A gene consists of multiple functional parts, including the coding region that specifies the protein to be made and regulatory elements that control the transcription of the coding region.

The reporter gene assay centers around fusing the putative regulatory elements to a reporter gene and monitoring the amount of the reporter protein expressed. Because reporter expression is under the control of the fused genetic elements, reporter expression is directly correlated with the activity of the regulatory elements. These elements could be promoters, enhancers or 5' or 3' untranslated regions (UTRs) that control either the transcription or translation events in the cell.

Denaturing high performance liquid chromatography (DHPLC) is a chromatographic analysis system that can be utilised to identify small scale mutations such as single nucleotide polymorphisms (SNPs), small insertions and small deletions. The DHPLC system displays high sensitivity for mutation detection at greater than 96%, so long as optimal denaturing conditions are selected, and so compares favourably to more traditional methods such as single strand conformational polymorphism and denaturing gradient gel electrophoresis, which have sensitivities of 82 and 85% respectively. 

The key steps require PCR amplification; homoduplex/heteroduplex formation; elution; detection and analysis. 

Can be used in noninvasive prenatal detection of chromosomal aneuploidy, trisomy 21, by measuring allelic ratios in maternal plasma samples using population SNPs 

It detects chromosome abnormalities for example translocations that may be difficult to see in traditional banding but also views all of the chromosomes at once which is important for considering complex karyotypes such as those seen in cancer cell lines. Additionally if all the chromosome are available to view at once then you do not need any a priori assumptions about where a chromosomal translocation may be.

It is not flawless. It is unable to detect rearrangements within a chromosome there is also been problems where the changes are very small and if they are from pericentric regions which can be very similar throughout the karyotype