strand-swtching and second-strand synthesis, ..

The kit alsocontains two optimized primers for reverse transcription and nuclease-freewater.

and second strand cDNA synthesis ..

The muscles used for locomotion in reside in the body wall. In the adult, there are 95 spindle shaped cells divided among four quadrants just underlying a basement membrane, hypodermis and cuticle. In each quadrant, the cells are arranged in interlocking pairs. In these muscle cells, myofilaments form a lattice that is restricted to a narrow zone of ~1.5 microns, just underlying the basement membrane and hypodermis. By polarized light microscopy, obvious striations are seen; bright (“birefringent”) A-bands alternate with dark I-bands; each I-band contains a row of dense bodies, which are the analogs of Z-discs of vertebrate striated muscle (). Because the striations lie at a slightly oblique angle with respect to the long axis of the worm, this muscle is called “obliquely striated”. Polarized light is also useful for evaluating the second largest set of muscles, those in the pharynx. Below we present a protocol for observing muscle using polarized light.

Incubate at 14°C for 12 hours and recover double-stranded cDNA by GENECLEAN II.

(second-strand cDNA synthesis, ..

The rcDNA genome is converted intocccDNA by cellular repair factors. Then, the cccDNA is transcribedto the pgRNA and subgenomic mRNAs (not shown). The mRNAs aretransported to the cytoplasm. The pgRNA is translated in thecytosol to form HBV core protein and the viral polymerase. Thesethree components assemble to form the core particle. The first(minus) DNA strand forms within the core particles via reversetranscription of the pgRNA to DNA; the pgRNA is degraded by viralRNase H as the plus strand is synthesized. (A) A3B inhibits thebinding of HnRnp K to the Enh II of HBV; (B) A3G may inhibit pgRNApackaging; (C) A3G renders HBV core protein-associated full-lengthpgRNA nuclease-sensitive; (D) A3G blocks DNA strand elongation andtargets a DNA-RNA hybrid. rcDNA, relaxed circular DNA; cccDNA,covalently closed circular DNA; pgRNA, pregenomic RNA; HBV,hepatitis B virus; APOBEC, apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like; HnRnp K, heterogeneous nuclearribonucleoprotein K; Enh II, enhancer II.

An anchored oligo-dT primer [d(T)23VN] forces the primer toanneal to the beginning of the polyA tail.

All members of the AID/APOBEC family possess one ortwo catalytic domains that deaminate cytidine in RNA and DNA. Thedeaminases mediate the hydrolytic removal of an amino group at theC4 position of a cytidine (C) or deoxycytidine (dC) generating auridine (U) or deoxyuridine (dU), respectively (). The presence of the enzyme in cellsproducing RNA virus results in C-to-U conversion of minus strandreverse transcripts and G-to-A in plus strand DNA. The binding tothe target DNA creates a U-G mismatch, which generates a C-to-Ttransition in minus strand DNA and a G-to-A transition in plusstrand DNA during general DNA replication without repair pathways() (). In humans, the family comprises 11members with distinct functions, including AID, APOBEC1 (A1),APOBEC2 (A2), APOBEC4 (A4) and APOBEC3 (A3) subgroups. The A3 groupconsists of seven proteins: A3A, A3B, A3C, A3DE, A3F, A3G and A3H(–). The seven A3 genes are arranged in atandem gene cluster on chromosome 22 in humans (). The presence of the AID/APOBECfamily is restricted to vertebrates. AID and A2 are likely to bethe ancestral members, while A1 and A3 are later evolutionaryarrivals (), A3s are restrictedto placental mammals, and their gene copy number isspecies-specific. For example, mice only possess a single A3 gene,pigs have two, sheep and cattle have three, cats have four, horseshave six and primates have at least seven A3 genes (). The rapid expansion of the A3 locusin humans indicates an important role in the host genome defenseagainst exogenous viruses and endogenous retroelements (–).The role of the AID/APOBEC family in the inhibition of viralinfection was initially described for HIV-1. Various studies haveshown that the genome of hepadnaviruses is hyperedited by cytidinedeaminases (–). Recent reports demonstrated that HBVDNA replication is restricted by A1, AID, A3A, A3B, A3C, A3G, A3F,but not A3DE (,,,–),specifically via the degradation of HBV cccDNA (), which was investigated in anexperimental setting through deaminase-independent and -dependentmechanisms (,).

Following second strand synthesis, transfer contents of 0.5 ml microfuge into a 1.5 ml RNase-free microfuge tube.


Synthesize first strand cDNA 2) Second strand cDNA 3) ..

Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM. At the end of the section we present a method for scanning EM (SEM) of worms. In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images ().

For the second-strand synthesis, the cDNA has to be eluted in 1 ..

Research on HBV reveals that the antiviral activityof A3G requires incorporation into assembling viral particles toinhibit reverse transcription. Factors required for incorporationof the antiviral deaminase protein, A3G into HBV nucleocapsidscontinues to be investigated. It has been demonstrated that A3G andA3C bind to the HBV core protein in immunoprecipitation experiments(,). Such binding is essential forreverse transcription following infection. The binding of A3G tothe HBV core protein was only indirectly demonstrated withcoexpression of RT and pgRNA, however, not with core protein alone(,). The results are consistent with thefindings of Nguyen () that A3G was specificallyincorporated into replication-competent HBV nucleocapsids byinteracting with viral RT and RNA packaging signals. However, byfluorescence resonance energy transfer (FRET) and acceptorphotobleaching experiments, Zhao () revealed that A3G directly binds tocore proteins. Similarly, direct interaction of HBV core proteinand A3A was confirmed by proximity ligation assay and FRETanalysis. Deletion analysis was used to confirm that the centralregion of the HBV core protein (between aa 77 and 149) was involvedin the interaction with A3A ().Additionally, the A3B, A3C and A3F enzymes were also found to beassociated with the HBV capsid by interaction with the core protein(). Similar to A3G, AID wasco-immunoprecipitated with the nucleocapsid core protein. Theassumption was made that AID formed a ribonucleopro-tein complexwith the HBV core proteins and RNA during nucleocapsid assembly inwhich AID deaminated cytosines of the viral RNA, including pgRNAand ssDNA ().

help for cDNA synthesis - Molecular Cloning - Protocol …

This priming method givesgood results when the amount of RNA is limiting (below 10 ng) and only oneparticular cDNA is desired.

Recommended primer concentration:
PRIMER-- Final conc.
OLIGO d(T)23VN -- -- 5 μM
RANDOM PRIMER MIX --6 μM
SPECIFIC PRIMER -- 0.1–1 μM