Question

1.describe southern and northern hybridization and the factors that influence it? 2.describe the clinical manifestations and pathogenesis of Staphylococcu aureus infections. How are they treated? 3.what is antiserum therapy, when is it used and what problems might arise from its use

Answer 1

1. The hybridization of a radioactive probe to filter-bound DNA or RNA is one of the most informative experiments that is performed in molecular genetics. Two basic types of hybridizations are possible.

·         Southern hybridization - hybridization of a probe to filter-bound DNA; the DNA is typically transferred to the filter from a gel

·         Northern hybridization - hybridization of a probe to filter bound RNA; the RNA is typically transferred to the filter from a gel

Probes are the primary tool used to identify complementary sequences of interest. Generally, the probe is a clone developed by inserting DNA into a vector. Most often these are plasmid clones.

·         Probe - a single-stranded nucleic acid that has been radiolabelled and is used to identify a complementary
nucleic acid sequence that is membrane bound

Factors influencing Nucleic acid hybridization:

Factors influencing sensitivity

The sensitivity of hybridization analysis is determined by how many labeled probe molecules attach to the target DNA. The greater the number of labeled probe molecules that anneal, the greater is the intensity of the hybridization signal. The specific activity is a measure of the incorporation of labeled nucleotides/probe. Modern labeling procedures as nick translation, random priming, PCR or in vitro RNA synthesis routinely provide probes with high specific activity.

The amount of target DNA is also essential and was already mentioned in chapter 1.1. The kind of labeling is another factor influencing sensitivity. Traditionally ³²P or ³⁵S labeled probes were used. Today nonradioactive probes are becoming increasingly popular as they have many advantages (see chapter 3).

Factors influencing specificity

The hybridization incubation is carried out in a high-salt solution that promotes base-pairing between the probe and target sequences. Hybridization is normally carried out below the Tm for the probe/target and the specificity of the experiment is the function of post-hybridization washes. The critical parameters are the ionic strength of the final wash solution and the temperature at which this wash is done.

The highly stringent wash conditions should destabilize all mismatched heteroduplexes so that hybridization signals are obtained only from sequences that are perfectly homologous to the probe.

Factors influencing hybrid stability and hybridization rate

A.) Hybrid stability

Ionic strength - Tm increases 16.6 °C for each 10-fold increase in monovalent cations between 0.01 and 0.4 M NaCl.

base composition - AT is less stable than GC base pairs in aqueous solutions containing NaCl

destabilizing agents - Each 1% formamide reduces the Tm by about 0.6 °C for a DNA-DNA hybrid. 6 M urea reduces the Tm by about 30 °C.

mismatched base pairs - Tm (melting temperature) is reduced by 1°C for each 1% of mismatching

B.) Hybridization rate

Temperature - Maximum rate occurs at 20-25°C below the Tm for DNA-DNA hybrids, 10-15°C below Tm for DNA-RNA hybrids

Ionic strength - Optimal hybridization rate at 1.5 M Na⁺

destabilizing agents - 50% formamide has no effect, but higher or lower concentrations reduce the hybridization rate

mismatched base pairs - each 10% of mismatching reduces the hybridization rate by a factor of two

duplex length - hybridization rate is directly proportional to duplex length

viscosity - increased viscosity increases the rate of membrane hybridization, 10% dextran sulfate increases rate by factor ten.

probe complexity - repetitive sequences increase the hybridization rate

base composition - little effect

pH - little effect between pH 5.0 and pH 9.0.

2. Staphylococcus infection:

Although there are many different primary clinical foci or manifestations of SAB, there are consistent patterns across cohorts. In several recent studies involving consecutive patients with either SAB (MSSA and MRSA) or only MRSA bacteremia common primary clinical foci or sources of infection are vascular catheter-related infections, SSTIs, pleuropulmonary infections, osteoarticular infections, and IE. These common primary clinical foci represent a subset of the common general clinical manifestations of S. aureus infections. However, a focus of infection is not found in ?25% of cases. As the clinical epidemiology of S. aureus infections changes, it is likely that the proportion of cases of SAB with these individual primary clinical foci will change. For example, reductions in catheter-related infections following improved infection control practices and implementation of central line bundles have resulted in catheter-related SAB contributing to a smaller fraction of all cases of SAB. Similarly, rates of SSTI-associated SAB are highest in communities with large numbers of cutaneous infections.

ignations have significant implications for the extent and type of diagnostic evaluation, duration of antibiotic treatment, and overall prognosis. A single-center study of 724 episodes of SAB defined complicated infection as one that resulted in attributable mortality, central nervous system (CNS) involvement, an embolic phenomenon, metastatic sites of infection, or recurrent infection within 12 weeks. Predictors of complicated SAB were community acquisition, positive examples include an increase in the incidence of USA300 community-associated MRSA (CA-MRSA) bacteremia with the widespread emergence of USA300 MRSA SSTIs as well as high incidences of both SSTI and SAB in indigenous populations.

SAB can be classified as “complicated” or “uncomplicated.” These dese follow-up blood cultures at 48 to 96 h, persistent fever at 72 h, and skin findings suggesting an acute systemic infection (petechiae, vasculitis, infarcts, ecchymoses, or pustules). The association between positive follow-up blood cultures and persistent fever with complicated SAB and subsequently poorer outcomes has been independently validated, as recently reviewed. The primary source of infection also predicts 30-day mortality, with higher mortality rates for bacteremia without a focus (22 to 48%), IE (25 to 60%), and pulmonary infections (39 to 67%), compared to lower rates for catheter-related bacteremia (7 to 21%), SSTIs (15 to 17%), and urinary tract infections (UTIs) (10%). Similar findings have recently been described in a pooled analysis of five prospective observational studies.

The management is done by beta-lactam antibiotic groups.

3. Antiserum therapy:

Antiserum, blood serum that contains specific antibodies against an infective organism or poisonous substance. Antiserums are produced in animals (e.g., horse, sheep, ox, rabbit) and man in response to infection, intoxication, or vaccination and may be used in another individual to confer immunity to a specific disease or to treat bites or stings of venomous animals. Antiserums from animals are most often used, but in persons allergic to animals, human antiserums have proved valuable.

Use:

Diagnostic and Treatment Purpose (Diphtheria, Tetanus).

Detection and Quantitation of Antigens

Analytical Reagents.

Tissue Typing

Problems:

Serological Reactions.

Hypersensitivity