Analyses of DNA, RNA and Protein
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Analyses of DNA, RNA and Protein What are the early discoveries and technological advances that revolutionized our ability to study human inherited disease? Structure of DNA Central dogma Restriction endonucleases Recombinant DNA technology Cloning Vectors Plasmids Double stranded circular DNA origin of replication selectable marker (antibiotic resistance) 1 or more restriction cutting sites can accommodate DNA fragments 5-10kbp Bacteriophage Lambda Large 25kbp double stranded molecule. Cosmids Can accommodate up to 50kbp of DNA YACs (yeast artificial chromosome Can accommodate up to 1000kbp of DNA BACs (Bacterial artificial chromosome) Can accommodate up to 600 kbp Human genomic plasmid library Also: Phage library YAC library BAC library cDNA library in plasmid Hybridization technology Molecular techniques for Analyzing DNA Southern blotting Detection of gene deletion by Southern blotting Southern blotting can be used to detect large alterations such as Deletions duplications Translocations Point mutations if they alter a restriction enzyme cutting site Quantitative Can be used to analyze large regions of DNA Polymerase Chain Reaction PCR analyses small regions of DNA Sequence of the region must be known to generate primers Not quantitative Advantage of PCR over Southern blotting Fast Sensitive Inexpensive PCR can be used to screen for unknown mutations in small regions of DNA using a variety of approaches, for example: Single strand conformation polymorphism analysis Single strand conformation polymorphism PCR can be used to assay for known mutations using numerous approaches for example: Multiplex analysis for small deletions Oligonucleotide hybridization Restriction endonuclease digestion if mutation alters a restriction site Allelic Discrimination by Real Time PCR Multiplex PCR analysis for DMD deletions CF mutation analysis using oligonucleotide probes PROBE Mutant Normal Homozygous normal Heterozygous parent Heterozygous parent Homozygous patient Niemann-Pick D mutation analysis (Bsu36I) 20 bp 203 bp Homozygous Heterozygous Homozygous normal mutant 223 bp 203 bp 20 bp Allelic Discrimination using Real Time PCR 1. A reporter (V or F) and a quencher (Q) are attached to the 5’ and 3’ end of a TaqMan probe. When both dyes are attached to the probe, reporter dye emission is quenched. 2. During each extesnion cycle, polymerase cleaves the reporter dye from the probe, enabling fluorescence 3. 2 probes with specificities that differ by a single base and have different fluorescent reporters can be used to descriminate alleles A G T T A G C C DNA fingerprinting Paternity testing Zygosity of twins DNA sequencing-Sanger method Detection of point mutations by direct sequencing of exons Next Generation Sequencing Cluster Generation DNA is fixed on a surface Amplification generates multiple fixed copies in close proximity Sequencing by Synthesis 4 fluorescently labeled nucleotides are presented to tens of millions of clusters on the flow cell surface in parallel During each cycle, one dNTP is incorporated and serves as a reversible terminator for polymerization. The fluorescent dye is imaged to identify which dNTP is incorporated then cleaved to allow addition of the next dNTP Software aligns sequences to reference samples and identifies variations. Next Generation Sequencing 1. Prepare Genomic DNA Sample 2. Attach DNA to surface Adapter DNA fragment DNA Dense lawn of primers Adapters Adapter Randomly fragment genomic DNA and ligate Bind single-stranded fragments randomly Adapters to both ends of the fragments to the inside surface of the flow cell channels 3. Bridge Amplification 4. Fragments Become Double Stranded Add unlabelled nucleotides and enzyme The enzyme incorporates nucleotides to build to initiate solid-phase bridge amplification double-stranded bridges on the solid-phase substrate 5. Denature the Double-Stranded Molecules 6. Complete Amplification Attached Attached Clusters Denaturation leaves single-stranded Several million dense clusters of Templates anchored to the substrate double-stranded DNA are generated in each channel of the flow cell 7. Determine First Base 8. Complete Amplification Laser The first sequencing cycle begins by adding After laser excitation, the emitted fluorescence four labeled reversible terminators, From each cluster is captured and the first primers and DNA polymerase base is identified 9. Determine Second Base 10. Image Second Chemistry Cycle Laser The next cycle repeats the incorporation of After laser excitation, the image is captured as four labeled reversible terminators, primers before and the identity of the second base is and DNA polymerase recorded 11. Sequencing Over Multiple 12. Align Data Chemistry Cycles The sequencing cycles are repeated to determine The data are aligned and compared to a the sequence of bases in a fragment, one base reference, and sequencing differences are at a time identified New Paradyme Sequence the entire genome and use software to generate data of interest Molecular techniques for analyzing RNA-gene expression Molecular techniques for analyzing RNA-gene expression Northern Blotting Reverse transcriptase PCR Microarray Reverse transcriptase PCR RT-PCR Microarray chips – gene expression profiles Gene expression profiles in lymphoma can be used for subclassification and stratification of treatment Realtime PCR-quantitative -used to assay for level of gene expression by using cDNA as a template -used to assay for residual disease in cancer patients after treatment -used to assay for level of gene expression by using cDNA as a template -used to assay for residual disease in cancer patients after treatment for example Molecular techniques for analysis of Protein Western blotting-gene expression Western blot Molecular cytogenetics Karyotype Interphase fluorescence in situ hybridiation (FISH) Trisomy 21 Metaphase FISH deletion Chromosome painting Probes for chromosomes 2 and 15 Spectral Karyotype (SKY) SKY showing deletion-insertion involving Chromsomes X and 14 Comparative genomic hybridization(CGH) CGH- a normal human metaphase spread was co-hybridized with normal and tumor DNA stained green and red respectively CGH Arrays Small overlapping chromosomal segments are bound to an array and hybridized with florescent tagged normal and patient DNA. This approach has very high resolution for detection of copy number Multiplex Ligation-dependent Probe Amplification (MLPA) Detection of aberrant copy number (DELETION OR DUPLICATION) of 45 genomic DNA sequences in one easy to perform, PCR based reaction. Technique: Denaturation Hybridization Ligation Amplification Each MLPA probe consists of two oligonucleoties, one consisting of a sequence specific for the forward PCR primer as well as sequence specific to a portion of the DNA region of interest. The second oligonucleotide consists of sequence specific for the reverse primer, an adjacent portion of the DNA region of interest and a stuffer sequence between the two, which is different in length for each probe. The two oligonucleotides will ligate together and amplify only if they bind side by side on the patient’s DNA. All probe ligation products are amplified by PCR using only one primer pair (as they have common primer sequences flanking the gene-specific sequences). The products of individual probes are distinguishable because the length of each amplification product is unique due to variations in the length of the stuffer sequence. The resulting amplification products are size separated and quantified by capillary electrophoresis.[8] MLPA output; trisomy 12 plus deletion 13q A 13q14.3 deletion ATM deletion B 13q14.3 deletion C Separation and quantification by capillary electrophoresis Each peak is the amplification product of a specific probe. Samples are compared to a control sample. A difference in relative peak height or peak area indicates a copy number change of the probe target sequence SNP Chip – can genotype thousands of SNPs on a single slide A large number (thousands) of single stranded oligos are chemically bound to a glass slide in groups of 4-each differing only in the last position by a single nucleotide (A,T,C or G) at a SNP site. The oligos on the chip are hybridized with fragmented, fluorescent tagged genomic DNA from a patient A computor reads the fluorescence and determines the genotype of the individual at that site Fast and cheap! Useful in mutation detection and for gene mapping Affymetrix Array 2,696,550 copy number markers 1,953,246 nonpolymorphic 743,304 SNP Can detect Copy number variation • Breakpoint determination Genotype variation Somatic mutations Loss of heterozygosity mosaicism Molecular cytogenetics-Summary Karyotyping Visualization of the whole genome Course resolution (>10Mb) Low yield Subjective Requires cultured cells (fresh tissue_ FISH Targeted genome only (locus specific) Medium resolution (>100kb) SKY Balance translocation screening Low resolution Multiplex ligation-dependent probe amplification (MLPA) Targeted genome only Need normal reference material Low resolution (up to 45 markers per regions of interest Array Comparative Genomic Hybridization (aCGH) Virtual karyotype High resolution SNP/CN Genotyping Arrays Virtual karyotyping for the whole genome High resolution (400kb-5kb) .