Mohd Nazri Ismail BSc in Biochemistry (2005) from UKM PhD in Molecular Biosciences (2011) from Imperial College

London Glycobiology, proteomics and mass spectrometry Doping Control Centre/ Pusat Kawalan Doping mnazri@dccusm.com

Techniques in Biotechnology BTT202

Dr. Mohd Nazri Ismail (first half) Dr. Zary Shariman Yahaya (2nd Half) Exam: 70% Lab report: 30% Course aim: Exposure towards understanding principles and techniques involved in genetic engineering as well as techniques in protein purification such as fermentation and chromatography My topics: Principles of genetic engineering, enzymology, cloning vectors, gene selection & screening, genomic library, and hybridisation techniques

BTT202 Principles of Genetic Engineering

The central dogma of molecular biology

Inside cell

DNA

DNA is the chemical basis of heredity, carrier of genetic information, organised into genes DNA directs the synthesis of RNA, which in turn regulates protein synthesis. The knowledge is important in defining normal cellular physiology and the pathophysiology of disease at the molecular level. First described in 1944 by Avery, MacLeod and McCarty

DNA STRUCTURE

A polymer of 2-deoxyriboneuclotides (CGAT) usually in double helix form, proposed by Watson & Crick in early 1950s based on their work and the work of Franklin & Wilkins

DNA units are polymerised by 3,5,phosphodiester bridges DNA exists in several double-helical structures, however B form is the dominant form under physiologic conditions.

Coiling and supercoiling

A-form

Right-handed double helix- B-form

Z-form

The 2 DNA strands are held together by hydrogen bonds between purine and pyrimidine bases The pairings between these bases are specific If A=T & G=C, so A + G (=/</>) T +C ??
A with T (a bonds) and G with C (3 bonds)

Melting temperature (Tm) =Denaturation of a double-stranded DNA molecule.

The base pairs

Size: Purines > Pyrimidines

Complementary A-T G-C

Nucleic acid base

Portion of a typical DNA Sequencing Electropherogram

http://www.hmc.psu.edu/core/oldsite/images/El ectropherogram.JPG

Complementary sequences

Chromosomes

DNA molecules are distributed among long linear structures called chromosomes Each chromosomes comprises a single DNA molecule associated with numerous proteins The number and size of the chromosomes are same in all cells of an organism but vary among different organisms Human has 23 pairs of chromosomes

DNA REPLICATION
Is the provision of progeny with the genetic information possessed by the parent. Must be complete and carried out with high fidelity to maintain genetic stability within the organism and species.

Involves many cellular functions and several verification procedures

Performed by DNA polymerase

DNA replication is semi-conservative

Nucleic acid synthesis

Addition of a nucleotide to the growing end of (3-OH) of a DNA strand

Deoxynucleotide triphosphate (dNTP)

DNA TRANSCRIPTION
Genetic information in DNA is expressed by synthesising mRNA as intermediates. The process is know as DNA transcription. The DNA sequence serves as template for the synthesis of mRNA. Performed by DNA-dependent RNA polymerase

Transcription the movie

RNA

Is a polymer of purine and pyrimidine ribonucleotides linked by 3,5phosphodiester bridges analogous to those in DNA Is the genetic material in some viruses and can be replicated In cells, RNA can be subdivided to mRNA, tRNA and rRNA with distinct functions

RNA STRUCTURE
Exists as a single strand, but may form a hairpin/ stem loop structure, thus acquiring double stranded characteristics. Contains uracil instead of thymine

mRNA
Functions as messengers conveying the information in a gene (DNA) to the proteinsynthesising machinery in the cytoplasm Very heterogeneous in size and stability

mRNA sequence serves as a template on which a specific sequence of amino acids is polymerised to form a specific protein molecule

rRNA

Is a key component in ribosomes Ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins form the mRNA templates rRNA is necessary for ribosomal assembly and binding of mRNA to ribosomes ant its translation. Mammalian ribosome contains 2 major subunit, small (40S) and large(60S).

tRNA

Serve as adapters for the translation of the information in the sequence of mRNA into specific amino acids There are at least 20 species of tRNA molecules in every cell and each of them correspond to at least one the 20 amino acids required for protein synthesis The nucleotide sequence of all tRNA allows extensive folding and intrastrand complementarity to generate a cloverleaflike secondary structure

Translation the movie

Dictionary of amino acid codes

Genetic engineering
Also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

Cloning

Is an asexual reproduction to obtain organisms that are genetically identical to one another and to parent. However, actual appearance and behaviour of the clones are still influenced by other factors. Gene cloning is the introduction of a foreign gene into a host so that it will be copied when the host replicates. Gene cloning involves isolation of the gene of interest, insertion into the host gene and isolation of the product (host and/or gene)
http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/clickandc lone.swf

Applications of cloning

Cloning animal models of disease

Reviving endangered or extinct specise

Pharming for drug production

Reproducing a deceased animal....and human?