Genetic testing 2
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How is sequencing done?
- The ssDNA to be sequenced is used as the template for DNA synthesis by DNA polymerase.
- A radioactive primer complementary to the 3′-end of the target DNA is added, along with the four deoxyribonucleoside triphosphates (dNTP).
- The sample is divided into four reaction tubes, and a small amount of one of the four dideoxyribonucleoside triphosphates (ddNTP) is added to each tube.
- Because it contains no 3′-hydroxyl group, incorporation of a ddNTP into a newly synthesized strand terminates its elongation at that point.
- The products of this reaction then consist of a mixture of DNA strands of different lengths, each terminating at a specific base.
- Separation of the various DNA products by size using polyacrylamide gel electrophoresis, followed by autoradiography, yields a pattern of bands from which the DNA base sequence can be read.
- The shorter the fragment, the farther it travels on the gel, with the shortest fragment representing that which was made first, that is, the 5′-end.
What are probes?
- Probe—a short, single-stranded piece of DNA, labeled with a radioisotope, such as 32P, or with a nonradioactive probe, such as biotin.
- The nucleotide sequence of a probe is complementary to the DNA of interest, called the target DNA. Probes are used to identify which clone of a library or which band on a gel contains the target DNA
How is Hybridization of a probe to DNA fragments performed?
- The utility of probes hinges on the phenomenon of hybridization (or annealing) in which a single-stranded sequence of a target DNA binds to a probe containing a complementary nucleotide sequence.
- ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane.
- The immobilized DNA strands are prevented from self-annealing, but are available for hybridization to an exogenous, single-stranded, radiolabeled DNA probe.
- The extent of hybridization is measured by the retention of radioactivity on the membrane.
- Excess probe molecules that do not hybridize are removed by washing the filter and, therefore, do not interfere
What are Synthetic oligonucleotide probes?
- If the sequence of all or part of the target DNA is known, single-stranded oligonucleotide probes of 20–30 nucleotides can be synthesized that are complementary to a small region of the gene of interest.
- If the sequence of the gene is unknown, the amino acid sequence of the protein—that is, the gene product—may be used to construct a probe. Short, ssDNA sequences (15–30 nucleotides) are synthesized, using the genetic code as a guide. Because of the degeneracy of the genetic code, it is necessary to synthesize several oligonucleotides
What are the differences between oligonucleotide probe and cDNA probe?
Oligonucleotides can be used to detect single-base changes in the sequence to which they are complementary. In contrast, cDNA probes contain many thousands of bases, and their binding to a target DNA with a single-base change is unaffected
a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of ...................................
When no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest what should be done?
In this case, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest
How can PCR be used for detecting mutation?
- 1. Direct sequencing
- 2. Indirect methods (restriction enzymes, allele-specific extension using fluorescently labeled nucleotides, also detection of changes in length)
What is allele-specific extension?
- Another approach for identifying mutations at a specific nucleotide position (say, a codon 12 mutation in the KRAS oncogene that converts glycine [GGT] to aspartic acid [GAT]) would be to add fluorescently labeled nucleotides C and T to the PCR mixture, which are complementary to either the wild-type (G) or mutant (A) sequence, respectively.
- Since these two nucleotides are labeled with different fluorophores, the fluorescence emitted by the resulting PCR product can be of one or another color, depending on whether a “C” or a “T” becomes incorporated in the process of primer extension
- The advantage of this “allele-specific extension” strategy is that it can detect the presence of mutant DNA even in heterogeneous mixtures of normal and abnormal cells (for example, in clinical specimens obtained from individuals with a suspected malignancy)
- Allele-specific PCR for mutation detection in a heterogeneous sample containing an admixture of normal and mutant DNA. Nucleotides complementary to the mutant and wild-type nucleotides at the queried base position are labeled with different fluorophores, such that incorporation into the resulting PCR product yields fluorescent signals of varying intensity based on the ratio of mutant to wild-type DNA present
differences in DNA length and PCR
- Diagnostic application of PCR and Southern blot analysis in fragile-X syndrome. With PCR the differences in the size of CGG repeats between normal and premutation give rise to products of different sizes and mobility. With a full mutation, the region between the primers is too large to be amplified by conventional PCR. In Southern blot analysis the DNA is cut by enzymes that flank the CGG repeat region, and is then probed with a complementary DNA that binds to the affected part of the gene. A single small band is seen in normal males, a band of higher molecular weight in males with premutation, and a very large (usually diffuse) band in those with the full mutation
How can Mutations that affect the length of DNA (e.g., deletions or expansions) also be detected by PCR analysis?
- Two primers that flank the region affected by trinucleotide repeats at the 5′ end of the FMR1 (example :fragile X) gene are used to amplify the intervening sequences.
- Because there are large differences in the number of repeats, the size of the PCR products obtained from the DNA of normal individuals, or those with premutation, is quite different.
- These size differences are revealed by differential migration of the amplified DNA products on a gel.
- At this point the full mutation cannot be detected by PCR analysis, because the affected segment of DNA is too large for conventional PCR.
- In such cases, a Southern blot analysis of genomic DNA must be performed
What are surrogate markers ?
- Detection of mutations by the PCR-based methods is possible only if the gene responsible for a genetic disorder is known and its sequence has been identified.
- In some diseases that have a genetic basis such approaches are not possible, either because the causal gene has not been identified or because the disease is multifactorial and no single gene is involved. In such cases, surrogate markers in the genome, also known as marker loci, can be used to localize the chromosomal regions of interest, on the basis of their linkage to one or more putative disease-causing genes
What are linkage analysis?
- Linkage analysis deals with assessing these marker loci in family members having the disease or trait of interest, with the assumption that marker loci very close to the disease allele are transmitted through pedigrees (linkage disequilibrium).With time it becomes possible to define a “disease haplotype” based on a panel of marker loci, all of which co-segregate with the putative disease allele. Eventually, linkage analysis facilitates localization and cloning of the disease allele.
- The marker loci used in linkage studies are naturally occurring variations in DNA sequences known as polymorphisms.
What are the marker loci used in linkage studies ?
What are the two types of genetic polymorphism that are most useful for linkage analysis?
- SNPs (including small insertion-deletion polymorphisms)
- Repeat-length polymorphisms known as minisatellite and microsatellite repeats.
What are common genetic variations?
- Short tandem repeat markers- Microsatellite markers
- Nucleotide substitutions and single nucleotide polymorphisms (SNPs)
- Insertion/deletion polymorphism
- Copy number variations (CNVs)
............................ represent the most abundant form of genetic variation
Single nucleotide substitutions
............................responsible for much of the heritable phenotypic variation observed in human populations
Single nucleotide substitutions
Lowest rate of base substitution is seen in.......
What are SNPs?
single-base pair changes that achieve a population frequency of at least 1 percent are referred to as single nucleotide polymorphisms (SNPs)
What are the mechanisms for Single-base substitutions resulting in SNPs or mutations?
Single-base slip mispairing during DNA replication and CpG-mediated cytosine deamination
What makes SNP suitable for analysis?
Because of their prevalence throughout the genome and relative stability, SNPs can be used in linkage analysis for identifying haplotypes associated with disease
What are repeat-length polymorphisms?
- Human DNA contains short repetitive sequences of DNA giving rise to what are called repeat-length polymorphisms.
- These polymorphisms are often subdivided on the basis of their length into microsatellite repeats and minisatellite repeats.
- Microsatellites are usually less than 1 kilobase and are characterized by a repeat size of 2 to 6 base pairs.
- Minisatellite repeats, by comparison, are larger (1–3 kilobases), and the repeat motif is usually 15 to 70 base pairs.
- It is important to note that the number of repeats, both in microsatellites and minisatellites, is extremely variable within a given population, and hence these stretches of DNA can be used quite effectively to establish genetic identity for linkage analysis.
- Microsatellites and the smaller minisatellites can be readily distinguished by utilizing PCR primers that flank the repeat region
How is genetic polymorphism produced in STR (short Tandem repeats)
- This polymorphism is generated by slip-mispairing during DNA replication, with formation of DNA loops and back priming of the leading strand.
- The longer the length of a segment of repetitive DNA, the more susceptible it is to slip-mispairing.
- Thus, mutation rates for STRs are substantially higher than for single-base pair mutations
What are the diseases associated with expansions of STR?
- Fragile X syndrome (due to expansion of a CGG repeat sequence in the 5'UTR of the FMR1 gene)
- Myotonic dystrophy (due to expansion of a CTG repeat in the 3'UTR of the DMPK gene)
- Huntington disease (due to CAG expansion within the coding region of the HD gene that encodes Huntingtin)
when the disease-associated gene is known, detection of the causative mutation by .................... is the method of choice
If the disease originates from several different mutations in a given gene (e.g., fibrillin-1), and gene sequencing is either not practical or negative but there is very strong clinical suspicion, ................... can be useful
What are other uses of microsatellite markers?
determining paternity and for criminal investigations
What are indels?
- Insertion/deletion (or indel) polymorphisms are polymorphic sites of gains or losses of one or more nucleotides.
- Most indels range in length between one and four nucleotides; the vast majority are one nucleotide
What are genomic disorders?
- Genomic disorders are diseases that result from the loss or gain of chromosomal/DNA material.
- The most common and better-delineated genomic disorders are divided in two main categories: those resulting from copy number losses (deletion syndromes) and copy number gains (duplication syndromes)
What are structural gene variations?
- sequence alterations spanning more than 1000 bases (one kilobase or kb)
- includes quantitative variations such as copy number variations (CNVs), sequence rearrangements (such as those observed among immunoglobulins), and other less common variations including chromosome inversions or translocations
What are CNV?
- CNVs, the most prevalent type of structural variation, are DNA segments spanning thousands to millions of bases whose copy number varies between different individuals.
- These submicroscopic genomic differences in the number of copies of one or more sections of DNA result in DNA gains or losses. Copy number gains can be the result of duplications, triplications, or even multiple copy number gains.
- Most deletions are one copy loss (heterozygous), but in some instances the loss can affect both copies (homozygous).
- most individuals carry an average of three large-scale CNVs
- CNV frequency is greatest in regions of segmental duplication (a 4- to 10-fold enrichment for CNVs), consistent with nonallelic homologous recombination as a primary mechanism for CNV mutation
When can CNVs be pathogenic?
if they involve a dose-sensitive gene(s) or if they influence genomic regions through regulatory elements
What is the mc mechanism for CNV?
- nonallelic homologous recombination
- Nonallelic homologous recombination (NAHR) typically involves the exchange of unequal amounts of genetic material during pairing between homologous chromosomes. Thus, the gene copy number is altered or hybrid genes are formed with novel properties
How are genomic disorders detected?
- Genomic disorders are typically detected by array comparative genomic hybridization (array CGH).
- Most laboratories confirm gains or losses detected on an array with an independent method such as fluorescence in situ hybridization (FISH), multiple ligation dependent probe amplification (MLPA), or quantitative PCR (Q-PCR
“Haploinsufficiency” (“haplo” = half) defines the concept where ..................................
loss or gain of one allele of a gene leads to abnormal protein production or function, thereby causing disease
Why linkage analyses of complex (multifactorial) disorders have been unsuccessful in identifying genes?
since conventional linkage studies lack the statistical power to detect variants with small effects and low penetrance, which are typical of the genes that contribute to complex disorders
Which method is used for identifying variants that are associated with low penetrance, small effects that contribute to complex multifactorial disorders?
What are the importance of identifying genetic variants in GWAS?
Such variants may themselves be causative, or may be in linkage disequilibrium with other genetic variants that are responsible for the increased risk
How is GWAS done?
- Using the publicly available “HapMap” data, the human genome is divided into “haplotypes” or regions of contiguous DNA inherited as a block, each identified by one or a few “tag” SNPs that identify the haplotype. In the example shown, locus 1 contains three haplotypes defined by different combinations of SNPs, where white signifies the more common “normal” sequence and each color designates a different SNP; thus, these haplotypes can be distinguished by assaying for only the blue and purple “tag” SNPs. Thereafter, high density SNP chips are constructed that contain these “tag” SNPs, in order to enable an unbiased genome-wide assessment of shared haplotypes between disease and control populations. Of note, “disease” refers to any defined phenotype, and could pertain to an actual disease entity like hypertension, or simply a quantitative trait like hair or eye color. Next, DNA obtained from the two cohorts is analyzed for overrepresented SNPs in the disease population (“cases”) versus the control samples—this is known as a case-control study
- . The most significant shared genomic regions of interest are then examined for candidate genes of interest—an example shown here in a search for loci associated with hypertension is angiotensinogen, a gene on chromosome 1 whose product regulates vascular smooth muscle tone. The final step is to perform a second case control study, this time using SNPs located within the gene of interest in order to confirm or refute the association with the trait, often in an independent population from the one in which the initial GWAS was conducted. In this example, individual SNPs within angiotensinogen gene are denoted as red vertical bars, and these SNPs will be tested in the second round of case-control study
What are some methods for identifying genomic alterations?
- Southern Blotting
- CGH array
How is Southern blotting done?
- Changes in the structure of specific loci can be detected by Southern blotting, which involves hybridization of radiolabeled sequence-specific probes to genomic DNA that has been first digested with a restriction enzyme and separated by gel electrophoresis. The probe usually detects one germ line band in normal individuals.
- Importantly, a normal DNA sample is required to compare the pattern of the DNA in question.
What are the steps in Southern blotting?
- First, DNA is extracted from cells, for example, a patient's leukocytes.
- Second, the DNA is cleaved into many fragments using a restriction enzyme.
- Third, the resulting fragments are separated on the basis of size by electrophoresis.
- [As the large fragments move more slowly than the smaller fragments, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.]
- The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis.If the original DNA represents the individual's entire genome, the enzymic digest contains a million or more fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA.
- If all the DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest.
The patterns observed on Southern blot analysis depend .........................................
both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments.
How is Fragile X syndrome diagnosed?
- (1) Southern blot analysis to measure the degree of methylation and the size of expansions in the large permutation or full mutation range
- (2) polymerase chain reaction (PCR) to identify normal alleles and discriminate small differences in the intermediate and premutation sizes, although a recently developed FMR1 mPCR assay may eliminate the need for Southern blot analysis.
- Southern blot analysis has the advantage of methylation determination, but is limited in its ability to distinguish normal from premutation allele sizes.
- PCR has the advantage of the need for less DNA, more sensitive determination of normal and premutation allele size, and less cost, but is limited in its ability to assess full mutations, as the PCR itself may selectively amplify shorter DNA segments.
What is the difference between utility of FISH and CGH?
- FISH requires prior knowledge of the one or few specific chromosomal regions suspected of being altered in the test sample.
- However, genomic abnormalities can also be detected without prior knowledge of what these aberrations may be, using a global strategy such as array CGH
How is array CGH performed?
- In array CGH the test DNA and a reference (normal) DNA are labeled with two different fluorescent dyes (most commonly Cy5 and Cy3, which fluoresce red and green, respectively).
- The differentially labeled samples are then hybridized to a glass slide spotted with DNA probes that span the human genome at regularly spaced intervals, and usually cover all 22 autosomes and the X chromosome.
- If the contributions of both samples are equal for a given chromosomal region (i.e., the test sample is diploid), then all spots on the array will fluoresce yellow (the result of an equal admixture of green and red dyes)
- In contrast, if the test sample shows an excess of DNA at any given chromosomal region (such as resulting from an amplification), there will be a corresponding excess of signal from the dye with which this sample was labeled.
- The reverse will be true in the event of a deletion, with an excess of the signal used for labeling the reference sample.
- Amplifications and deletions in the test sample can now be significantly better localized, often down to a few thousand base pairs.
- Array CGH is regularly performed in cases of mental retardation–developmental delay of unknown etiology or in children with dysmorphic features with negative karyotypes.
CNVs can be detected using
What are the disadvantages of Array CGH technology?
- There are usually many CNVs observed when comparing any two genomes encompassing millions of bases of DNA. Deciding whether a specific change is a benign polymorphism or a critical disease-causing duplication or deletion can be difficult.
- Another limitation of existing array CGH platforms is that they cannot detect balanced translocations, since there is a rearrangement but no genetic material is gained or lost.
What is epigenetic?
- Epigenetics is defined as the study of heritable chemical modification of DNA or chromatin that does not alter the DNA sequence itself.
- Examples of such modification include the methylation of DNA, and the methylation and acetylation of histones
- regulation of tissue-specific gene expression, X chromosome inactivation, and imprinting
Gene expression frequently correlates with the level of ..................................................................................................
methylation of DNA, usually of cytosines specifically in the CG dinucleotide-rich promoter regions known as CpG islands
analysis of promoter methylation is used for diagnosis of...................
Methylation analysis is used for diagnosis of ............and.........
What are the methods used for detection of DNA methylation?
- One common approach is to treat genomic DNA with sodium bisulfite, a chemical that converts unmethylated cytosines to uracil, while methylated cytosines are protected from modification.
- An assay termed methylation-specific PCR uses two PCR primer sets to analyze single DNA loci: one to detect a DNA sequence with unmethylated cytosines (which are converted to uracils after bisulfite treatment) and the other to detect DNA sequences with methylated cytosines (which remain cytosines after bisulfite treatment).
What is chromatine immunoprecipitation?
New techniques are based on the ability to detect histone modifications such as methylation and acetylation (which, like DNA methylation, are important regulators of gene expression) by using antibodies against specifically modified histones. Such antibodies can be used to pull down bound DNA sequences, a method termed chromatin immunoprecipitation (ChIP). These pulled-down sequences can be amplified and analyzed by hybridizing to microarrays (“ChIP on Chip”) or sequencing (“ChIP-Seq”) to map epigenetically modified genes throughout the genome
........................is the method of choice for detection of minimal residual disease in patients with chronic myeloid leukemia
PCR performed on cDNA
Why is PCR performed on cDNA the method of choice for detection of minimal residual disease in patients with chronic myeloid leukemia?
- Because most translocations occur in scattered locations within particular introns, which can be very large, beyond the capacity of conventional PCR amplification.
- Since introns are removed by splicing during the formation of mRNA, PCR analysis is possible if RNA is first converted to cDNA by reverse transcriptase
How can gene expression be studied?
- 1. mRNA
- Northern blots
- 2. Proteins
- ELISA, Western Blot, Proteomics
What is DNA microarray?
- DNA microarrays contain thousands of immobilized DNA sequences organized in an area no larger than a microscope slide.
- These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping), or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time.
- For genotyping analysis, the cellular sample is genomic DNA.
- For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag
- This mixture is then exposed to a gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene.
- The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample.
- DNA microarrays are often used to determine the differing patterns of gene expression in two different types of cell—for example, normal and cancer cells.
Approximately .....% of CNVs and ....% of SNP involve gene-coding sequences
There is a significant over-representation of certain gene families in regions affected by CNVs; these include genes involved in the ............................................................
immune system and in the nervous system
What are the general features of miRNA?
- miRNAs, unlike other RNAs, do not encode proteins but instead inhibit gene expression.
- Silencing of gene expression by miRNA is preserved in all living forms from plants to humans and therefore must be a fundamental mechanism of gene regulation.
- Because of their profound influence on gene regulation, miRNAs are assuming central importance in understanding normal developmental pathways, as well as pathologic conditions, such as cancer.
How is miRNA processed?
- About 5% of the human genome
- Transcription of miRNA genes produces primary miRNA transcripts, which is processed within the nucleus to form another structure, called pre-miRNA
- With the help of specific transporter proteins (exportin), pre-miRNA is exported to the cytoplasm. Additional “cutting” by an enzyme, appropriately called Dicer, generates mature miRNAs that are about 21 to 30 nucleotides in length (hence the name “micro”)
- At this stage the miRNA is still double-stranded. Next, the miRNA unwinds, and single strands of this duplex are incorporated into a multiprotein complex called RNA-induced silencing complex (RISC).
- Base-pairing between the miRNA strand and its target messenger RNA (mRNA) directs the RISC to either cause mRNA cleavage or repress its translation.
- In this way, the gene from which the target mRNA was derived is silenced (at a post-transcriptional level)
- given miRNA can silence many target genes.
- siRNAs works in a manner quite similar to that of miRNA. Unlike miRNA, however, siRNA precursors are introduced by investigators into the cell.
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