IL 15: Molecular Methods

(B) ATTCGCAATCA Read 5' to 3', bottom to top

(A) ACTAACGCTTA (B) ATTCGCAATCA (C) ATCTATCGATC (D) GCCCTTTAAAA (E) AAAATTTCCCG

(C) Triplet repeat expansion Symptoms of mytonic dystrophy which is a result of trinucleotide repeat expansion

(A) Gene duplication (B) Gene deletion (C) Triplet repeat expansion (D) Increased SNPs in the disease state (E) Differences in restriction fragment length polymorphisms (RFLPs)

A patient has been diagnosed with a particular form of cancer. Appropriate treatment of this cancer, however, requires knowledge of which molecular markers are being expressed by the tumor as compared to normal cells of the same tissue. This is most easily accomplished by which of the following techniques? (A) Southern blot (B) Southwestern blot (C) MicroRNA analysis (D) Microarray analysis (E) ELISAs

(D) Microarray analysis Test the control and cancer tissue

PCR Method

1) Denaturation step: The reaction is heated (~95 deg) to "melt" the DNA template into single-stranded DNA molecules. 3) Annealing step: The reaction is cooled (~60 deg), allowing the primers to anneal to the complementary regions flanking the sequence of interest. 3) Elongation step: DNA synthesis at 72 deg using the primers to start chain extension. This is done using a thermostable DNA polymerase isolated from bacteria that live in hot springs or thermal vents.

The most common systems involve bacterial hosts or eukaryotic cell lines that undergo three basic steps:

1) transfecting the gene of interest into the producing cell 2) expressing large quantities of the product of interest 3) harvesting/purifying the drug

Automated Sequencing Method

1. All four reactions are done in one tube. 2. The tube contains the four deoxynucleotides (dATP, dCTP, dGTP, dTTP). -Each dideoxynucleotide is fluorescently tagged with a different-color dye (ddATP, ddCTP, ddGTP, or ddTTP). 3. The mixture is separated by size using capillary electrophoresis, and a fluorometer detects the labeled chains. 4. The sequence can be determined by reading the colors as they pass through the detector.

Steps for chromosomal microarray:

1. Create/obtain a DNA microarray that contains multiple single-stranded DNA sequences from various regions throughout the genome (dozens to thousands) 2. Isolate DNA multiple samples for comparison (example - fetal sample and reference sample) 3. Amply PCR and label each of the samples (reference-green versus fetal-red) 4. Cohybridize equal amounts of the fluorescently labeled DNAs on the array and scan the microarray, and quantitate the signal to quantitate the relative amount of each of the DNAs binding the probe 5. Interpretation based on the example in figure Yellow - equal quantity of genomic DNA from control and test sample Red - increased quantity of genomic DNA in the test sample (segment duplicated) Green - decreased quantity of genomic DNA in the test sample (segment deleted)

Micro-satellite Analysis Method

1. DNA is extracted 2. Microsatellite region is amplified 3. Size of PCR product assessed

ASO Hybridization Method

1. DNA samples are PCR amplified to enrich the sequence of interest and integrate a label into the product 2. Short probes (15-20 nucleotides in length) spotted onto a membrane or other substance. -Probes that bind to normal sequences -Probes that bind to disease-causing variants 3. The labeled DNA sample is applied to the probes and allowed to hybridize -The short length of the probe prevents the hybridization if there is a single-base pair mismatch between the probe and the DNA in the sample -This can detect if an individual is homozygous or heterozygous for a given SNP 4. Note: this technology is only effective if one knows where to look for the mutation and the specific modification

Steps for gene expression microarray

1. Dozens to thousands of single-stranded DNA sequences or probes from the coding strand of numerous genes dotted on the array (like a mega ASO) 2. Isolate and purify mRNA from TWO ( or more) samples for comparison − One sample is the control − One is the test sample (an individual with the disease, pharmacologically treated, a different stage of development, etc.) 3. Reverse transcribe and amplify using PCR − A different fluorescent label is incorporated into each of the mRNAs − Example: cDNA from a normal tissue (green) cDNA from a malignant tissue (red) 4. Cohybridize equal amounts of the fluorescently labeled cDNAs on the array 5. Scan the microarray and quantitate the signal to quantitate the relative amount of each of the cDNAs binding the probe Interpretation based on the example in figure Yellow - equal quantity of transcript from control and test sample Red - increased number of transcripts in the test sample (gene up-regulated) Green - decreased number of transcripts in the test sample (gene down-regulated)

Next Generation Sequencing Method

1. Extract DNA and generate a library of thousands to millions of small fragments based on the template DNA 2. Amplification of the library 3. The fragments are sequenced in parallel using one of several methods. 4. The sequences of the fragments are then reassembled to yield the whole sequence. 5. This technique requires areas to be sequenced several times. This is called coverage. The accuracy of the sequence is often described by the number of times a section is sequenced

Multiplex PCR Method

1. Multiple primer sets are created that span several exons and in some cases intron 2. The primers generate products of different sizes 3. The products are separated on a gel and visualized

The essential difference between traditional ASO and genotyping microarrays

1. The matrix that the probe binds (membrane versus glass or bead) 2. Density/number of different alleles routinely tested. -ASO involves the dotting of a few probes onto the surface. -SNP arrays can test for hundreds of different sequences. 3. The most significant limitation is that one needs to know the precise structure of the gene and create oligos to detect the disease-causing mutation. -This can be challenging when there can be hundreds of different alleles associated with the disease.

Explain the method of CRISPR

1. They create a small piece of RNA with a short "guide" sequence that attaches to a specific target sequence in a cell's DNA. This is called the gRNA. 2. This guide RNA also attaches to the Cas9 enzyme, which guides the enzyme to the specific DNA sequence to be targeted. 3. The Cas9 enzyme cuts the dsDNA and forms a double-stranded break at the targeted location. 4. The cell then repairs the DSB using nonhomologous end joining (NHEJ), which usually deletes a few nucleotides. -The result of this NHEJ is the silencing of the edited gene by frameshift mutations of inducing nonsense mutations. 5. Once the DNA is cut, researchers can use the cell's own DNA repair machinery to -Delete genetic material (gene knock-out) -Add new genetic material (gene knock-in) -Modify existing sequences using a customized DNA sequence (gene editing) Note If genes are inserted it can also result in a homologous event where no DNA is loss

RT-PCR Method

1. Uses reverse transcriptase and primer to anneal and extend the desired mRNA sequence. 2. The reverse transcriptase and primer will anneal to the mRNA sequence and transcribe a complementary DNA strand (cDNA) if the mRNA is present. 3. Multiple cycles of PCR are then used to amplify target sequences in the cDNA. Note: Oligonucleotide T primer is generally used since it can bind to the poly A tail

Antibody microarray

A complex mixture of proteins to measure protein specificities and protein expression levels of the proteins in the mixture. In this technique, a library of antibodies is arrayed on a glass microscope slide. The array is then probed with a protein solution.

B. Additional analysis is required to determine if the child would be affected. PCR detects the size of the DNA. The results indicate the child has a normal and abnormal reading. The 250bp was inherited from the mothering, indicating a recessive allele. However, PCR does NOT detect point-mutations. Therefore, we do not know if the child inherited the normal allele from dad or the one with a mutation. ASO or micrroarray test would need to be done to detect.

A couple had a child with Hurler syndrome, a rare autosomal recessive disorder caused by the inability to degrade mucopolysaccharides. Genetic analysis revealed that the mother has a 200 bp deletion within exon 2 of her α-l-Iduronidase gene while the father has a missense mutation in exon 2. During her subsequent pregnancy, she undergoes chorionic villus sampling and DNA testing to evaluate the risk of recurrence. PCR is used to amplify exon 2 in the fetus. The results of this reaction are: Which of the following would be the best interpretation of this information? A. A northern blot of RNA from this child is likely to be normal. B. Additional analysis is required to determine if the child would be affected. C. The child couldn't be a carrier of the disease. D. The child will have Hurlers disease. E. The child won't have Hurlers disease

Whole Exome Sequencing

A strategy of sequencing only the coding regions of a genome Less complex, and faster However, misses alteration in the non-coding sequence which can be important in disease progression

Determine the number of copies of a given gene within a genome. A. Southern blot analysis B. Northern blot analysis C. Restriction fragment length polymorphism (RFLP) analysis D. Transfection E. Polymerase chain reaction (PCR)

A. Southern blot analysis

What test would you use to detect point-mutations of DNA?

ASO or microarray

Eukaryotic Expression Systems

Able to undergo post-translational modifications Possess cellular machinery that allows glycosylation and improved protein folding Time-consuming, lower yield, more costly

RT-PCR

Allows the detection and quantification of mRNA in a sample (cells, tissues).

How is Western Blotting used for HIV, Hepatitis C, Lyme Disease, etc

Allows the identification of auto-antibodies if the virus is present

Track a disease locus within an affected family. A. Southern blot analysis B. Northern blot analysis C. Restriction fragment length polymorphism (RFLP) analysis D. Transfection E. Polymerase chain reaction (PCR)

C. Restriction fragment length polymorphism (RFLP) analysis

PCR

Copies minute amounts of DNA a million or a billion-fold using multiple heating and cooling cycles This exponential amplification occurs using primers (short synthetic DNA fragments) that bind known sequences in the original template DNA that flank the region to be amplified.

32-year-old pregnant woman with a history of deep venous thrombosis associated with oral contraceptive use is seen for her first prenatal visit. Her mother also had an episode of deep vein thrombosis during pregnancy. DNA from the woman is tested for a potential mutation in the factor V gene known to cause this condition. The DNA sequence to be amplified by PCR is shown below. The dashed line indicates the internal sequence where the potential mutation is located. Which answer choice represents the pair of primers that should be used in the PCR? TCCTGAGC------------------------------AAATGTGT A. AGGACTCG and TTTACACA B. GCTCAGGA and AAA TGTGT C. GCTCAGGA and ACACA TTT D. TCCTGAGC and ACACATTT E. TCCTGAGC and TTTACACA

D. TCCTGAGC and ACACATTT Need a complimentary template to match the 3' end of the starting strand, allowing us to synthesize 5' to 3' On the new strand made, its new primer would be complementary to the 3' end which is the 5' of the original strand

Southern Blotting

DNA is digested/cut with a restriction enzyme, separated by gel electrophoresis, transferred onto a specialized blotting membrane, and hybridized with a labeled complementary DNA probe for the target sequence

Recombinant DNA

DNA produced by combining DNA from different sources

Chromosomal microarray

DNA-based method of genetic analysis can identify clinically significant chromosome abnormalities below the resolution of conventional chromosome analysis.

What is the purpose of Southern Blotting

Detect significant (large) changes in the DNA, such as translocations, insertions, or deletions Analysis of Restriction Fragment Length Polymorphisms

What is the purpose of DNA sequencing?

Determines the order of the four bases—adenine, guanine, cytosine, and thymine— in a strand of DNA

OpsII is a hypothetical type II restriction endonuclease with properties similar to those of other restriction enzymes discussed in class. Which DNA sequence shown below is most likely to be the recognition sequence of OpsII? A. GGAGGA B. GGAAGG C. GAGGTG D. GGACCT E. GGATCC

E. GGATCC Restriction enzymes read palindromic sequences. Read in the same direction 5' to 3' as 3' to 5' to 3'

What are restriction enzymes?

Endonucleases that recognize specific base sequences and cut the sugar-phosphate backbones of the DNA strand Recognition sequences are generally palindromic

What is the purpose of Northern Blotting

Examine specific gene expression levels across tissues, developmental stage, in response to drugs, or under pathological conditions Can also measure the actual size of mRNAs to look for splice variants or aberrant splicing patterns

Use of Microsatellite Analysis

Genetic mapping and linkage analysis to trace inheritance of DNA Assess uniparental heterodisomy DNA fingerprinting Measure microsatellite instability to identify individual at increased risk certain cancers due to errors in DNA mismatch repair mechanisms

Single Nucleotide Polymorphism (SNP) analysis

Identifies SNPs among alleles Measuring predisposition to disease, identifying drug candidates, evaluating germline mutations in individuals, assessing the loss of heterozygosity, or genetic linkage analysis

Microsatellite Analysis

Identifying a specific individual through PCR amplification of microsatellite DNA. Microsatellites are tandem repeated sequences, where the repeating unit is 2 to 7 nucleotides long

Blotting

Labeled probes are used to identify target seq in DNA, RNA or proteins that have been separated by gel electrophoresis

What does the CMA look out for?

Looks for clinically significant duplicated or deleted chromosomal material and copy number variants These chromosome aberrations include microdeletions, microduplications, most abnormalities of chromosome number (trisomy, monosomy), and most unbalanced rearrangements of chromosome structure (translocations, etc.) It can be designed to detect excessive homozygosity (uniparental disomy) and triploidy (69, XXY).

Microbial pathogen detection

Microarrays can detect and identify pathogens (bacteria, viruses, fungi, and parasites). This can be very important in diagnosing and treating infectious diseases, checking for specific organisms in food, and preventing or tracking infectious diseases.

Allele-Specific Oligonucleotide (ASO) Hybridization. Limitations?

PCR-based technique used in genetic tests to detect KNOWN single nucleotide polymorphisms (SNPs), short deletions, or insertions.

Western Blotting

Protein samples are separated by electrophoresis and transferred to blotting membranes. The target proteins are visualized using labeled antibodies Note: Protein is denatured

Gene Expression Microarray

Quantify the concentration of a gene's mRNA transcripts in a cell

Northern Blotting

RNA (or mRNA) is extracted from cells, separated by electrophoresis, transferred to a membrane, and hybridized with a labeled complementary nucleotide probe for the target sequence Note: RNA is NOT cut by restriction enzymes

Microarray analysis

Rely on high-density probes being deposited and bound to specific sites on the surface of a "chip" (similar to ASO) 1. Each dot holds a single-stranded DNA sequence (probe) or a monoclonal antibody 2. The immobilized probes can bind the fluorescently labeled targets in the test samples. 3. Because the array depends on the complementary binding of the unknown sequences or proteins, it will enable parallel analysis of hundreds to thousands of known targets at once.

Trinucleotide repeat disorders such as Fragile X can result in methylation, silencing a gene (FMR1). Therefore, why is Southern blot added to the assessment of Fragile X syndrome when the amplification in the FMR1 gene can be quantitated by PCR?

Restriction enzymes in Southern Blotting can NOT cut in methylated sites, therefore, these regions can easily be detected and provide additional information about the disorder

What test would you use to detect large deletions of DNA?

Southern Blot or PCR

Dideoxy Termination Method

Takes advantage of DNA polymerization requiring a 3' OH group to halt elongation 1. The template DNA is denatured to form a single strand 2. A short primer (in orange) is annealed to the target sequence 3. 4 reactions were done in parallel. -Each reaction included the four deoxynucleotides (dATP, dCTP, dGTP, dTTP). -Each reaction included a small amount of a single dideoxynucleotide (ddATP, ddCTP, ddGTP, or ddTTP). -When incorporated, the dideoxynucleotide (marked by an *) prevented elongation of the DNA strand. 4. In each tube, the random incorporation of the dideoxynucleotide results in products of different lengths, and each tube is run in a different well. 5. The DNA chains are separated by size using electrophoresis. The sequence is determined by reading the gel from the smallest fragment (the 5'end) to the largest fragment (the 3' end). NOTE: Smallest fragments travel longer distance in gel electrophoresis so you read from the bottom , up

D. Point mutation that abolishes the RNA splice site Patient has a traditional sized mRNA and a larger sized mRNA. Therefore, only D would result in that

The results show a northern-blot of a patient who is being tested for familial hypercholesterolemia. Based on the blot, which of the following mutation is MOST LIKELY causing the disease in the patient? A. Frameshift at codon 138 B. Missense mutation preventing the processing of the protein C. Nucleotide change that alters the stop codon used in translation D. Point mutation that abolishes the RNA splice site E. Promoter mutation that leads to increased transcription

Explain how RNAs could be constructed to guide a Cas nuclease to any DNA sequence

The so-called guide RNA allows the system to target a single, specific sequence increasing the likelihood of the appropriate site being edited and decreasing (but not eliminating) editing of other sites in the genome. Effectively disrupts the targeted gene If a DNA template is added to the system, a new sequence can be inserted into the desired spot

Multiplex PCR

Using multiple primer sets to simultaneously amplify multiple target sequences. Multiplex PCR analysis can identify the loss or addition of one or more exons in the gene of interest.

What is RFLP?

Variation of the length of restriction fragments due to the addition or deletion of restrictive sites Various lengths results in different migrations in electrophoresis Used for inheritance markers (AA, aa, Aa)

Bacterial Expression Systems

Widely used because of ease of handling and high yield Unable to glycosylate proteins which can impact solubility Difficulty generating multidomain eukaryotic proteins due to issues with post-translational modifications and molecular folding Lower cost

Several genetic diseases are caused by the expansion of trinucleotide repeats through

anticipation Note: PCR can effectively quantitate the number of repeats in an individual using primers flanking the unstable site

When using PCR to identify trinucleotide repeats, Premutation alleles (carriers) would appear to have

do not exhibit the trait, but they could have a child with the trait if expands through anticipation to a full mutation

The CRISPR/Cas9 system has the capacity to

edit specific genes

When using PCR to identify trinucleotide repeats, Full mutation alleles would appear to have

expansions that lead to the disease phenotype

When using PCR to identify trinucleotide repeats, a Wild-type allele (normal) would appear to have

generally, the number of repeats does not expand

ASO technology has largely been replaced by

genotyping microarrays/SNP arrays

CMA does NOT detect

point mutations, small duplications, and deletions of DNA sequences with a single gene (for example, trinucleotide repeats) and balanced chromosomal rearrangements (balanced translocations, inversions).

CRISPR/Cas9 was developed from a

primitive defense mechanism used by bacteria to disable viruses that have caused infections in the past The bacterium takes a piece of the viral genome and inserts it into its own. − If it encounters the virus again, the bacterium generates RNA segments from the CRISPR array that binds to and destroys the matching viral genome. − Researchers adapted this immune defense system to edit DNA

Whole Genome Sequencing

process that determines the DNA sequence of an entire genome (both nuclear and mitochondria)