BIO203 Final Term Merged Slides

MINI-PRIMER PCR

Mini Primer PCR uses a thermostable polymerase (S-Tbr) that can extend from short primers as short as or nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.PCR that can extend from short primers

ADVANTAGES AND LIMITATIONS OF PCR

ADVANTAGES

Specific, Simple, rapid, relatively inexpensive, Amplifies from low quantities Works on damaged DNA Sensitive Flexible, Contamination risk, Primer complexities, Primer-binding site complexities, Amplifies rare species, Detection methods

APPLICATIONS OF PCR

APPLICATIONS

Detection of Infectious diseases: AIDS, TB, CMV, H1N1, etc Viral, Bacterial and fungal infections, Diagnosis of latent viruses. Forensic applications: DNA finger printing Detection of Mutations: Inherited disorders carriers Track DNA abnormalities . Prenatal diagnosis of genetic disorders. Detection of pathogens Pre-natal diagnosis DNA fingerprinting Gene therapy Mutation screening. Drug discovery, Classification of organisms Genotyping, Molecular Archaeology Molecular Epidemiology Molecular Ecology, Bioinformatics Genomic cloning . Site-directed mutagenesis Gene expression studies

Polymerase chain reaction (PCR) PCR-Gene cloning and

expression

PCR has been used in gene cloning and screening of genomic libraries

Applications of PCR PCR has widespread applications in various fields of life sciences including genetic engineering, medical, forensic, agriculture, environment etc

Polymerase chain reaction (PCR) PCR-Medicine PCR has major impact on medicine especially in the field of clinical microbiology or diagnosis Molecular tools have also allowed to perform prenatal genetic diagnosis\

Polymerase chain reaction (PCR

PCR-Forensic sciences: Forensic science is the application of scientific procedures to solve criminal and legal matters. Molecular methods are used to established the filiations of a person or to obtain evidence from minimal samples of saliva, semen or other tissues PCR-DNA profiling: DNA profiling or DNA fingerprinting is a forensic technique used to identify individuals by characteristics of their DNA

Polymerase chain reaction (PCR)-PCR-Agricultural sciences and environment

PCR has also facilitated research in detection of pathogens in plants, animals and environment

Polymerase chain reaction (PCR PCR-Agricultural sciences and environment

PCR has also facilitated research in detection of pathogens in plants, animals and environment

Cutting DNA molecules Types of restriction and modification (R-M) system

Type I, Type II, Type III, Type IIs Type I: Type I systems were the first to be characterized from E. coli K12 The active enzyme consists of two restriction subunit, two modification subunit and one recognition subunit. Type I systems are of little value for gene manipulation Type II: Most of the useful R-M system is Type II. Type II enzymes recognize a defined sequence and cut within it Type III: Type III enzymes have symmetrical recognition sequences but otherwise resemble type I systems and are of little value Type Iis: Type IIs systems have similar cofactors and structure to type II but restriction occurs at a distance from recognition site that limits their usefulness. Cutting DNA molecules--Nomenclature : A suitable system was proposed by Smith and Nathans (1973). The species name of host organisms is identified by the first letter of genus and first two letters of specific epithet E. coli = Eco, H. influenzae = Hin

Strain identification is written as EcoK . In case, host strain has several restriction and modification systems, these are identified by roman numerals, for example, in case of H. influenzae, HindI, HindII, HindIII etc. All restriction enzymes have general name endonuclease R and modificationmethylase M followed by the system name, for example, in case of H. influenzae. R. HindIII or M. HindIII Examples of restriction endonuclease nomenclature

Cutting DNA molecules

Target sites Type II endonucleases recognize and cleave DNA within particular sequences of four to eight nucleotides which have a twofold axis of rotational symmetry i.e. referred as palindromes 5’-GAATTC-3’ 5’-CTTAAG-3’ 5’-G A A T T C-3’ 3’-C T T A A G-5’ 5’-G/ A A* T T C-3’ 3’-C T T A* A/ G-5’ 5’-G 5’- A A T T C-3’ 3’-C T T A A-5’ G-5’ Single stranded breaks by EcoR1

Number and size of restriction fragments The number and size of fragments generated by restriction enzyme depend on the frequency of occurrence of the target site in the DNA to be cut. Four base recognition site occurs every 44 (256) bp Six base recognition site occurs every 46 (4096) bp Eight base recognition site occurs 48 (65,536) bp Average fragment size (bp) produced by different enzymes

Cutting DNA molecules

Summary of restriction endonucleases: Type II restriction enzymes are heavily responsible for the current explosion in the field of gene manipulation in that they are essential in forming recombinant DNA. .

Joining DNA molecules-- DNA modifying enzymes Nucleases, DNA Polymerase, Reverse transcriptases, DNA ligases Nucleases: Nucleases or DNases are the enzymes that degrade DNA. Two broad classes of nucleases i). Exonucleases ii). Endonucleases DNA Polymerase: An enzyme that catalyzes template-dependent synthesis of DNA Reverse transcriptases: An RNA-directed DNA polymerase in retroviruses; capable of making DNA complementary to an RNA DNA ligase: An enzyme that creates a phosphodiester bond between the 3’ end of one DNA segment and the 5’ of another Methods of joining DNA fragments : Mainly three methods are used for joining DNA in vitro i). Joining covalently annealed cohesive ends by DNA ligase ii). Joining blunt-ended fragments by DNA ligase from phage T4 infected E. coli

iii). The third utilizes the enzyme terminal deoxynucleotidyltransferase to synthesize homopolymeric 3’ single stranded tails at the ends of fragments DNA ligase: E. coli and phage T4 encode enzyme, DNA ligase, which seals single stranded nicks between adjacent nucleotides in a duplex DNA chain DNA ligase to create covalent recombinant DNA:” DNA ligase can repair the nicks produced after the association of cohesive ends of DNA strands Use of DNA ligase to create a covalent DNA recombinant. Alkaline Phosphatase: An enzyme responsible for removing phosphate groups from many types of molecules including DNA. Treatment of linearized plasmid vector DNA with alkaline phosphatase to remove 5’-terminal phosphate groups will prevent recircularization and plasmid dimer formation. Application of alkaline phosphatase

Blunt end ligation via linker molecules: Joining DNA fragments with cohesive ends by DNA ligase is a relatively efficient process T4 DNA ligase has been used to joint blunt-ended DNA molecules.

Adaptors: Chemically synthesized adaptor molecules which have a preformed cohesive end can be used to insert blunt ended foreign DNA into vector. The main difference between linker and adaptor is that former having blunt ends while later have one cohesive end. SOUTHERN BLOTTING: Southern Blotting is named after its inventor, the British Biologist Edwin Southern (1975). BLOTTING: Southern Blotting is named after its inventor, the British Biologist Edwin Southern (1975). OTHER BLOTTING METHODS: Other blotting methods with similar principles, but using protein or RNA, have been named in reference to Edwin Southern's name after. SOUTHERN BLOTTING Identify DNA sequence (gene) of interest. Identified DNA may be a small piece of DNA or a mutation

SOUTHERN BLOTTING PROCEDURE: DNA is extracted from cells, leukocytes. DNA is cleaved into many fragments by restriction enzyme (e.g, BamH1, EcoR1 etc) . The resulting fragments are separated on the basis of size by electrophoresis. The DNA fragments are denatured and

transferred to nitrocellulose membrane for analysis. The labeled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets. Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe. Radioactive probes enable autoradiographic detection. Southern blot is used to detect the presence of a particular piece of DNA in a sample by a molecular probe.

SOUTHERN BLOTTING PROBES –PROBES: Labeled material to detect a target. For DNA: 20-30 nucleotides, complementary to a region in the gene or DNA. RADIOACTIVE PROBE - P32: Sensitive Relatively cheap Hazardous Radioactive waste disposal regulations should be followed NON-RADIOACTIVE PROBE –BIOTIN: Sensitive Relatively expensive.. HYBRIDIZATION OF PROBES: The binding between single stranded labeled probe to a complementary nucleotide sequence on the target DNA.. PROBES: Labeled material to detect complementary region in the gene or DNA.