Question

1. Describe the principles behind and the applications of the following:

a) Southern blotting

b) cDNA synthesis

c) CRISPR-Cas9 gene editing

d) S1 Mapping or Primer Extension

e) Sanger Sequencing of DNA

f) Site-directed mutagenesis

j) cDNA cloning

k) Western Blotting

l) Restriction Enzymes

please answer them all because its including in one question !

Answer 1

1.

a) Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is mainly based on the principle of separation of DNA fragments by gel electrophoresis. It is identified by labelled probe hybridization. Southern blotting is useful for analysis of repetitive sequences because multiple similar sequences in the genome can be analyzed by a single probe. Its quantitative results reflect the amounts of digested and undigested DNA molecules.

b) Using mRNA as a template, reverse transcriptase produces its complementary DNA (cDNA) based on the pairing of RNA base pairs. cDNA can be obtained from both prokaryotes and eukaryotes. The main advantage of cDNA is we can store the cDNA that was reverse transcribed from the RNA.

c) A single RNA consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein and it binds to a recombinant form of Cas9 protein that has DNA endonuclease activity. This third-generation gene editing technique CRISPR-Cas9 has higher targeting efficiency and accuracy, lower cytotoxicity and lower costs for being easier for vector design and manipulation and less off target effect.

d) Primer extension provides the same type of information as S1 mapping. However, the primer extension is unaffected by splice sites and in case where only a genomic probe is available and an intervening splice site prevents S1 mapping of the start site, primer extension offers a useful alternative. Coming to advantages, primer extension offers additional advantages than the S-1 mapping. A genomic clone of the target RNA is not required; only 30 - 50 bases of sequence need be known to generate the primer and additionally probe preparation is easier, because the primer is single stranded.

e) Sanger sequencing of DNA is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Advantages of sangers sequencing of DNA includes cost-efficient, simultaneous interrogation of more than 100 genes at a time. Finding novel variants by increasing the number of targets sequenced per run. Analysis of samples with low-input DNA. Sequencing of whole genomes, especially microbial genomes.

f) Site directed mutagenesis is a method to create specific, targeted changes in double stranded plasmid DNA. It is a method to create specific, targeted changes in double stranded plasmid DNA. It is used to select or screen for mutations that have a desired property and to introduce or remove restriction endonuclease sites or tags. It is also used to study the changes in protein activity that occur as a result of the DNA manipulation.

j) The main principle of cDNA cloning is mRNA population isolated from a specific developmental stage should contain mRNAs specific for any protein expressed during that stage. Advantages includes there are no introns. Eukaryote genes commonly contain introns and these are removed after mRNA synthesis so cDNA contains no introns. This means that a cDNA copy of a gene can be isolated as a single, intron-free fragment.

k) Western blotting is a rapid and sensitive assay for the detection and characterization of proteins. It is also known as protein blotting or Immunoblotting. It is mainly based on the principle of immunochromatography where the proteins are separated into polyacrylamide gel according to their molecular weight. Western blotting is commonly used for verification of protein production after cloning. It is also used in medical diagnostics. For example, it is used in the HIV test or BSE-Test. The confirmatory HIV test employs western blotting to detect anti-HIV antibody in a human serum sample.

L) Restriction enzymes are the nucleases which can cleave the sugar-phosphate backbone of DNA that found in bacteria. Nowadays restriction enzymes are an indispensable tool in the field of biotechnology and main advantage of these restriction enzymes are that they offer the means to very precisely cut through a double strand of DNA. As per recent analysis, over 19,000 restrictive enzymeshave been identified to-date.