"Biochemisitry1a. We can find specific mutations or set of mutations that leads to differential expression ofmutated genes by following methods,1. Enzymatic mutation detection methodThis method uses an enzyme resolvase , endo vii, cloned from the bacteriophage t4.this enzymehas high specificity to detect insertions, deletions and base substitutions mutations ormismatches. first the pcr is used to amplify the normal and mutant allele of target sequence, thesepcr primers are labeled with fam(6-carboxyfluoroscein amidite) a blue fluorescent dye andreverse primer with tet(4,7,2,7-tetra chloro-6-carboxy fluoroscein)amidite, a green fluorescentdye.Denaturation and renaturion of normal and mutant allele in a mixture is done, as a resultmismatch heteroduplexes will form approximately 50 percent of the time, each base pair formstwo mismatch. the endo vii enzymes now scan the double stranded DNA until it detectsstructural distortion, either a bubble caused by single base pair mutations or a hetero duplex loopformed by hybridization of a wild type allele with a mutant allele containing an insertion ordeletion. The enzyme cleaves with in 6 base pair on 3”side of mutation forming two shorterfragments, one blue other green. The DNA sequence is analyzed on automated DNA sequenceand mobility of each fragment is evaluated.2.two dimensional gene scanning methodTwo dimension gene scanning method is a high resolution system for detection of mutationalvariants in multiple genes. This method is based on combination of extensive polymerase chainreaction and two dimensional DNA electrophoresis a customized computer program was used todesign optimal primer combination for pcr amplification, denaturing gel electrophoresis andDNA fragments distribution id 2d gel, this test permits the detections of mutations in codingregions as well as splice site mutations.3. Protein truncation test The protein truncation test (ptt) provides a rare exception, targeting mutations that generateshortened proteins, mainly premature translation termination. Ptt has several attractivecharacteristics, including pinpointing the site of a mutation, good sensitivity, a low false-positiverate, and, more importantly, the near-exclusive highlighting of disease-causing mutations. Inaddition, ptt facilitated the detection of a new mutation type, i.e., a sequence change generating ahypermutable region surfacing in the RNA. This process uses RNA as template and scans formutations at the protein level. Protein truncation test is composed of four steps1. Isolation of nucleic acid either genomic dna, total RNA or poly (a) rna.2. Amplification of specific gene of interest3. In vitro transcription and translation of the product of the amplification reaction4. Detection of translation of the products by autoradiography.The shorter protein products of the mutant alleles are easily distinguished form the full lengthprotein product of mutant allele.Diagram in the attachment4.single stranded conformation polymorphism analysisThis is a widely used screening method that identify different genomic mutations in largenumber of samples,this techenique detecte sequence variations due to point mutations or othermutations through electrophoretic mobility diference,as DNA that contains a sequence mutationhas a measurable mobility difference compare to wild type DNA when subjected to nondenaturing conditions.This process consists of 4 steps1.digestion of genomic DNA with restriction endonucleases2.denaturation in an alkaline solution.3.electrophoresis on a neutral polyacrylamide gel.4.transfer to a nylon membrane.5.hybridisation with either DNA fragments or RNA copies synthesized on each strand as probes.Diagram in the attachment5.dhplc Denaturing high pressure liquid chromatography is new technique is very useful screening largenumber of sample of mutations, best for germ line mutations, this method involves heteroduplexformation between wild type and mutated DNA strands to identify mutations. hetro duplexmolecules are separated from homoduplex molecule by ion pair, reverse liquid chromatographyon a special matrix with partial heat denaturation of the DNA strandsB,Techniques involving quantification of RNA species1. Northern blottingThe amount of RNA species can be quantified by measuring the quantity of RNA by northernblotting which give size and sequence information about the RNA molecule.In this method a sample of RNA is separated on an agaraose gel and hybridise to a radioactiveRNA probe that is complementary to target sequencer. the radio labeled RNA is then detected byan autoradiograph.2. reverse transcriptase-real time quantitative pcrAnother technique for quantification of mrna is rt-qpcr, in this method reverse transcriptiongenerates a DNA template from the mrna, this single stranded template is called cdna. The DNAtemplate is then amplified by rt-qpcr in a quantitative step during which fluorescence emitted bylabeled hybridization probes or intercalating dyes changes as DNA amplification processproceeds. with the standard curve qpcr can produce an absolute measurement of the number ofcopies of original rna.3. Hybridisation micro arrayThese techniques consist of single array or chip that contain probes to determine transcript levelfor every known gene in the genome thus help in quantification of RNA species. 4. Serial analysis of gene expression and RNA sequencing are tag based technologies which canprovide a relative measurement of the cellular concentration of different rna.2. Answer C DNA is produced from a mature m RNA of euakrayotic cell with the use of enzyme known asreverse transcriptase.Steps in c DNA construction1.m RNA is obtained and purified from rest of the rna.2.the m RNA purified is tagged with oligo d t nucleotide as a complementary primer which bindsto pol a tail of m RNA creating a free 3”oh end that can be extended by reverse transcriptase tocreate a complementary DNA strand. now the m RNA is removed by rnase enzyme leaving asingle stranded c dna. this single stranded c DNA is converted to double stranded DNA with thehelp of DNA polymerase.Diagram in the attachmentThe sanger method, also referred to as dideoxynucleotide sequencing or chain terminationsequencing, is based on the use of dideoxynucleotide (ddntp) in addition to the normalnucleotides (dntp) found in c DNA. Dideoxynucleotide are essentially the same as nucleotidesexcept they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (oh). Thesemodified nucleotides, when integrated into a cdna sequence, prevent the addition of furthernucleotides thus stop the elongation of the DNA chain. This occurs because a phosphodiesterbond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus theDNA chain is terminated.Steps in sanger method of cdna sequencinngThe region of cdna to be sequenced is amplified and then denatured to produce single strandeddna. A sequencing primer is annealed to the single stranded dna. Dideoxynucleotide chain termination DNA sequencing then takes advantage of the fact that agrowing chain of nucleotides, extending in the 5’ to 3’ direction, will terminate if, instead of aconventional deoxynucleotide, a 2’3’ dideoxynucleotide becomes incorporated. By performingfour separate reactions, each containing a DNA polymerase and a small amount of one of the four dideoxynucleotides in addition to all four deoxynucleotides, four separate sets of chain- terminated fragments can be produced. Following the replication/termination step, these chain terminated fragments will remain boundto the single stranded DNA molecule which has acted as a template. By heating these partiallydouble stranded molecules and adding a denaturing agent such as formamide, the single strandedchain termination molecules can be released from their template and separated using highresolution denaturing gel electrophoresis. The sequence of the original region of cdna is then finally deduced by examining the relativepositions of the dideoxynucleotide chain termination products in the four lanes of the denaturinggel.3 answerC DNA amplification1. Smart-pcr amplification of cdna is the process which starts with the conversion of mrna tocdna using mmlv-rt, mutated in the rnase h domain, in the first strand synthesis reaction . Thisenzyme possesses reverse transcriptase activity, terminal deoxynucleotidyl transferase (tdt)activity, and dna-dependent DNA polymerase activity. During first strand synthesis, an anchoredoligo-d(t) primer (3’ cdna synthesis primer) anneals to the 5’end of poly(a) tails.. The 5’-end ofthis 3’ cdna synthesis primer contains a defined sequence that serves as a pcr primer target site inthe subsequent amplification step. When the first strand has been extended to the end of an mrnatemplate, the tdt activity of the reverse transcriptase adds several un-templated deoxycytosine(d(c)) residues to the 3’-end of first strand cdnas (in a fashion analogous to the addition of singleun-templated d(a) residues to the end of pcr products generated with taq DNA polymerase). The5’cdna synthesis primer contains three guanosine residues at its 3’ end that anneal to these un- templated d(c) “tails”And then serve as a template for the dna-dependent DNA polymerase activity of the mmlv-rt.This “switching mechanism at RNA termini” (smart) allows for mmlv-rt-mediated 3’-extensionof all first strand cdna products to include a defined, contiguous sequence copied from the 5’cdnasynthesis primer. The terminal transferase and template switching activities occur during the firststrand cdna synthesis. Regions of sequence in the 5’cdna synthesis primer and the 3’cdnasynthesis primer are identical, allowing for uniform amplification of all first strand cdna with a single pcr primer.2.ssh-pcr relies on the principles of DNA hybridization. Cdnas prepared from the two mrnapopulations being compared are first digested with a four base cutting restriction enzyme togenerate short blunt-ended fragments which are more favorable to the suppressive pcr action.One of the cdna population which is believed to contain an overrepresentation of some unknowngenes, is denoted as “tester”. The tester cdna is split to two aliquots and each is ligated to adifferent adaptor . The other cdna population is denoted as “driver” and is mixed with the twoadaptor-ligated tester cdnas separately. The two mixtures are allowed to undergo denaturationand renaturation. in these complexes, the driver is in excess and hybridizations do not go tocompletion. Sequences over-represented in the tester cdna pool remain single stranded andsequences common to both tester and driver reanneal as they nucleate more frequently due totheir higher relative concentrations. In a second hybridization step, the single stranded testersequences remaining in the two first hybridization mixes, which bear different adaptor sequencesat theirends, are allowed to fully re-anneal in the presence of additional denatured driver cdna. Inthis second hybridization, there is further subtraction of sequences common to both tester and "