Question

09. Describe specific molecular techniques that detect DNA, mRNA or polypeptide. Identify specific probes used for the detection of the corresponding macromolecules. Is there a technique available to identify a protein that interacts with DNA sequence? (10 points) Q10A. Differentiate between two types of DNA sequencing methodologies, sequencing by chain termination and next generation sequencing. (5 points)

Answer 1

(@) The key to detection of specific nucleic acid sequences is base pairing between complementary strands of RNA OF DNA. At high temperatures (e.g.- 90°C to 100°c), complementary Stromds of DNA separate (denature), yielding single-stranded molecules. If such denatured DNA strands are then incubated under appropiate conditions (e.g-65°), they will renature to form double-stranded molecules are dictated by complementary base pairing - a process nucleic acid hybridization. DNA → Southern hybridization detection of specific genes called in cellulare DNA. Tue DNA is digested by restriction endonuclease and the digested DNA fragments are separated by gel electrophoresis gel then overlaid with a mitro cellulose filter or nylon membrane, to which the DNA fragments are transferred (blotted) to yield replica of the gel. filter is then incubated with a radio labeled probe , which hybridizes to the DNA fragments that cout all the complementary sequence. these fragments can by exposure of the filter to x-ray film. The be visualized

determining SDS - Polyacrylamide gel electrophoresis (SDS-PAGE) MRNA- Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression Four populare methods : Northern blot analysis, nuclease poolection assays (NPA), in situ hybridization, and reverse transcription - polymerase chain reaction CRT-PCR). Norrtern analysis remains the standard for detection aud quantification of mRNA levels despite the advent of a reusitive techniques. The most compelling of these is that it to the easiest method for transcript size, alternatively spliced transcripts and multigene family members. Here, total cellular RNAs are extracted and fractionated according to size by gel ele chrophoresis ender deuatwing conditions. The RNA is then transferred to as membrane, crosslinked and hybri- -dized with a labeled probe. Non isotopic or high specific activity na diolabelled probes can be used including random- - primed, nick-hromolated. PCR-generated DNA probes, in vitro transcribed RNA probes and oligonucleotides, CDNA with only partial homology used as probes. Antibodies can be used in a variety of ways proferis m cell extracts. Two comonon methods are immunoblotting (Westem blotting) and immunoprecipitation. Proleurs extracted are first separated according to size by gel electrophoresis. Because profecies have different wohapes and charges, however, this process requires a modification of méthods used for electophersis of nucleic acid instead can be Protein detection - to detect

The DNA bindi Protein molecules denature getting overall negative charge. Then all proteins migrate toward the positive electrode - there nates of migration determined only by size. The prokies are transferred to a filter, which is there allowed to react with antibiodies against the protein of interest. antibody bound to the filter can be detected by Nosious methods, thereby identifying the profein aganiet which the antibody is targeted. Molecular technique to detect DNA interaction with protein- ng proteins are understood previously Electrophoretic mobility Shift assay (EMSA). + doue in vitro purified protein or a cresce extract of protein is incubated with labelled DNA. Some per proteins involued in branscriptional regulation or other varied function bind weakly to DNA making their isolation and identification difficult Another method → curomatin immunoprecipitation (chip) Chip defines spatial and temporal relationship of a particular protein-DNA interaction. Chip assay can also be used to analyze binding of transcription factors, transcription co-factors, DNA replication factors and DNA repair proteins where in

10A) SANGER Fast, cost-effective sequenci- •Higher sequencing depth enables TARGETED SEQUENCING NGS Benefits D (1-20 targets). • Familiar workflow. ng for low numbers of targets higher sensitivity (down to 1%) lo Higher discovery power (ability to identify novel variants) •Higher mutation resolutions (identifying smallest to largest hentations). • More data produced with the same DNA input amount. • Higher bample throughput. Challenges Low sensitivity Chinit of • Less cost effective for detection w 15-20%) lewer number of targets (220) 1. Low discovery power • Time consuming for lower Not as cost effective for numbers of targets (1-20 targets) (720 targels) • Low scalability due to increasing sample input requirements. higher number of targets