Principles of Genetic Enginering 2012

7/30/2019 Principles of Genetic Enginering 2012

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Mohd Nazri Ismail

BSc in Biochemistry (2005) from UKM

PhD in Molecular Biosciences (2011) fromImperial College London

Glycobiology, proteomics and mass spectrometry

Doping Control Centre/ Pusat Kawalan Doping

[email protected]


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Techniques in Biotechnology

BTT202Dr. 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


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BTT202

Principles of Genetic Engineering


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The central dogma of molecular biology


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Inside cell
http://../Mohd%20Nazri%20Ismail/Desktop/Inside%20cell.wmvhttp://../Mohd%20Nazri%20Ismail/Desktop/Inside%20cell.wmv


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DNA

DNA is the chemical basis of heredity, carrier ofgenetic information, organised into genes

DNA directs the synthesis of RNA, which in turnregulates protein synthesis.

The knowledge is important in defining normalcellular physiology and the pathophysiology ofdisease at the molecular level.

First described in 1944 by Avery, MacLeod andMcCarty


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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 workof Franklin & Wilkins


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DNA units arepolymerised by 3,5,-phosphodiesterbridges

DNA exists in

several double-helicalstructures, howeverB form is thedominant form under

physiologicconditions.


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Coiling and supercoiling

Right-handed double

helix- B-form

A-formZ-form


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The 2 DNA strands are held together by hydrogenbonds between purine and pyrimidine bases A with T (a bonds) and G with C (3 bonds)

The pairings between these bases are specific

If A=T & G=C, soA + G (=/) T +C ??

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


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The base pairs

Nucleic acid base

Size: Purines > Pyrimidines

Complementary

A-T

G-C


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Portion of a typical DNA SequencingElectropherogram

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

ectropherogram.JPG


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Complementary sequences


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Chromosomes

DNA molecules are distributed among longlinear structures called chromosomes

Each chromosomes comprises a single DNAmolecule associated with numerous proteins

The number and size of the chromosomesare same in all cells of an organism but varyamong different organisms

Human has 23 pairs of chromosomes


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DNA REPLICATION

Is the provision of progenywith the genetic informationpossessed by the parent.

Must be complete and carried

out with high fidelity tomaintain genetic stability withinthe organism and species.

Involves many cellular functionsand several verificationprocedures

Performed by DNApolymerase


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DNA replication is

semi-conservative


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Nucleic acid synthesis

Addition of a nucleotide to the growing

end of (3-OH) of a DNA strand

Deoxynucleotide triphosphate (dNTP)


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DNA TRANSCRIPTION

Genetic information in DNA is expressedby 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 RNApolymerase

Transcription the movie
http://../Mohd%20Nazri%20Ismail/Desktop/TranscriptionAdvancedMovHQ.movhttp://../Mohd%20Nazri%20Ismail/Desktop/TranscriptionAdvancedMovHQ.mov


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RNA Is a polymer of purine and pyrimidine ribonucleotides linked by 3,5-

phosphodiester 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


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RNA STRUCTURE

Exists as a single strand, butmay form a hairpin/ stem

loop structure, thus

acquiring double stranded

characteristics.

Contains uracil instead of

thymine


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mRNA

Functions as messengers conveying theinformation in a gene (DNA) to the protein-

synthesising machinery in the cytoplasm

Very heterogeneous in size and stability


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mRNA sequence serves as a template on

which a specific sequence of amino acids is

polymerised to form a specific proteinmolecule


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rRNA

Is a key component in ribosomes

Ribosome is a cytoplasmic nucleoproteinstructure 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 itstranslation.

Mammalian ribosome contains 2 major subunit,small (40S) and large(60S).


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tRNA

Serve as adapters for the translation of theinformation in the sequence of mRNA intospecific amino acids

There are at least 20 species of tRNAmolecules in every cell and each of themcorrespond to at least one the 20 aminoacids required for protein synthesis

The nucleotide sequence of all tRNA allowsextensive folding and intrastrandcomplementarity to generate a cloverleaf-like secondary structure


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Translation the movie
http://../Mohd%20Nazri%20Ismail/Desktop/DNAi_translation_vo2-sm.wmvhttp://../Mohd%20Nazri%20Ismail/Desktop/DNAi_translation_vo2-sm.wmv


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Dictionary of amino acid codes


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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.


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Cloning

Is an asexual reproduction to obtain organisms that aregenetically 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 ahost 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
http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/clickandclone.swfhttp://learn.genetics.utah.edu/content/tech/cloning/clickandclone/clickandclone.swfhttp://learn.genetics.utah.edu/content/tech/cloning/clickandclone/clickandclone.swfhttp://learn.genetics.utah.edu/content/tech/cloning/clickandclone/clickandclone.swf


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Applications of cloning


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Cloning animal models of

disease


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Reviving endangered or extinct specise


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Pharming for drug production


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Reproducing a deceased animal....and human?


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Text of Principles of Genetic Enginering 2012