Molecular Biotechnology

C G V ipin Shankar

Fundamentals of Molecular Biotechnology
 Revolutionary scientific discipline based on the ability to

transfer specific units of genetic information from one organism to another.

 Relies on the techniques of genetic engineering -

Recombinant DNA Technology (RDT)

Aims of Molecular Biotechnology
 To create a useful product or a commercial

process, by choreographing the various Molecular Waltzes of life, taking place largely inside the cells.  Biotechnology is a term, that covers various techniques for using the properties of living things to provide products & services.

Biotechnology operates at the molecular levels of life, where the seemingly solid boundaries between species disappear. Down among those molecules there lies little difference between a man and a bacterium. What biotechnology does is to choreograph the complex dances among the molecules that ultimately make every living thing what it is.

The Molecular Biotechnology Revolution
 Molecular biotechnology is only the current

chapter in a story that began long time ago.  In the modern context neither bread making or wine fermentation, nor genetic alteration by selective breeding, or plant cloning by grafting, or the use of microbial products in fermentation; are considered as Biotechnology

The Molecular Biotechnology Revolution…
 What is new about modern biotechnology is not

the principle of using various organisms but the techniques for doing so.  Biotechnology has produced so many applications very rapidly.

In the beginning
 1665 : Robert Hooke

and the Onion peel – ‘The Cells’.  1675 : Anton Van Leeuwenhoek and the microscopes – shape & size of various microscopic particles.

As the years passed….
 1839 : Matthias Schleiden

& Theodore Schwann – ‘The Cell Theory’.  1859 : Darwin the Voyager – ‘On the origin of species by natural selection’.  1866 : Mendel the Monk - ‘Genetics and Mendelism’.

As the years passed…
 1869 : Johann Miescher – Discovery of DNA  1900 : Rediscovery of Mendelism  1902 : Walter Sutton – Genes present on

Chromosomes.  1910 : Morgan and the fruit flies.  1928 : Griffith – ‘The transforming element’.

As the years passed…

As the years passed…
 1941 : Beedle & Tatum-

‘one gene one enzyme’.  1944 : Oswald Avery – DNA the genetic carrier.  1953 : Watson and Crick – Structure of DNA

As the years passed…
 1967 : Har Gobind Khorana & Marshal

Nirenberg – The Genetic Code.

Molecular Biotechnology Now
Microbiology Biochemistry Immunol ogy Genetics Chemical Engineeri ng

Molecular Biology

Cell Biology

Molecular Biotechnology

Crops

Drugs

Vaccines

Diagnostics

Livestock

Basic concept of RDT
 Techniques for  Identifying (locating),  Isolating,  Altering &  Studying DNA segments

D (daring) N (nucleotide) A (adventures)

Techniques for handling DNA
 Preparation of pure samples of DNA.  Cutting DNA molecules.  Analysis of DNA fragment sizes.  Joining DNA molecules together.  Introduction of DNA into host cells.  Identification of cells that contain recombinant

DNA molecule.

Vehicles for carrying DNA
 Plasmids.  Phages.  Viruses.

Plasmids
 Circular DNA molecules of independent      

existence in the bacterial cell. Carry one or more gene. Carry selectable markers. Have at least one origin of replication May exist as episomes Vary in size and copy number. Various incompatibility groups exist.

Plasmids…
 Classified into  Fertility of F Plasmids.  Resistance or R Plasmids.  Col Plasmids.  Degradative Plasmids.  Virulence Plasmids.

Bacteriophages
DNA hijackers.  Viruses infecting bacteria.

Have protective capsids.  Lytic & Lysogenic life cycles.

Viruses
 Absence of plasmids in higher cells limit use of

plasmids and phages as vectors.  Have considerable potential as cloning vehicles in animal cells.

Purification of DNA from living cells
 Three distinct kinds of DNA usually isolated  Total cell DNA.  Pure Plasmid DNA.  Phage DNA.

Preparation of Total Cell DNA
 The cells are grown and harvested.  The cells are broken open to release their

contents.  The cell extract is treated to remove all components except the DNA.  The resulting DNA solution in concentrated.

Preparation of cell extract
 Physical methods

 Enzymatic method
 

Mechanical force
 

Grinding Centrifugation

Lysozyme Lysozyme + EDTA

 Chemical methods
  

Ethylene di-amine tetra acetate (EDTA). Detergents – SDS Cetyl tri-methyl ammonium bromide (CTAB).

Purification of DNA from crude extract
 Deproteinization  1:1 mixture of phenol and chloroform  Protease treatment (Pronase, Proteinase K)  Ribonuclease treatment to degrade RNA  Anion exchange resins.

Concentration of DNA
 Ethanol precipitation.  In the presence of monovalent cations like Na+, at temperature – 20 oC or less.  Sticks to glass rod.  Leaves short chain and monomeric nucleic acids

in solution.  Added advantage of removing RNA traces.

Measurement of DNA concentration
 Ultra violet absorbance spectrophotometry.  Measured at 260 nm.  A 260 of 1.0 corresponds to 50 µg of ds DNA.  Purity of DNA can also be assayed.  Pure sample has (A 260 / A280) = 1.8.

Ratios of less than 1.8 indicate contaminations by proteins or phenol.

Preparation of Plasmid DNA
 Similar to Genomic DNA isolation.  Physical differences between genomic and

plasmid DNA exploited for separation.
  

Differences in size and conformation. Plasmids are very small compared to genomic DNA. After extraction plasmids remain circular while genomic DNA is broken and occur in linear conformation.

Separation on the basis of size
 Cells lysed under specific conditions –  EDTA + Lysozyme treatment in presence of sucrose.  Sucrose prevents cell burst.  Spheroblasts – partially wall-less cells that retain genomic DNA, are formed.  Cell lysis induced by non-ionic detergents (Triton X100).  Debris removed by centrifugation.  Clear lysate consisting entirely of plasmid DNA left.

Separation on the basis of conformation
 Three different conformations  Supercoiled DNA.  Covalently closed-circular DNA.  Open circular DNA.  Plasmids exist as supercoils.  Can be separated by –  Alkaline denaturation.  EtBr- CsCl density gradient centrifuagation.

Alkaline denaturation
 At a pH range of 12.0 – 12.5, non-supercoiled DNA is   

denatured while supercoiled DNA is not. Cell extract (Preferably clear lysate) is adjusted to this pH using sodium hydroxide. Causes unwinding of double helix and denaturation of non-supercoiled DNA. Addition of acid to the mixture causes denatured DNA to aggregate to form tangled masses- removed by centrifugation. Supercoiled Plasmid DNA remains in solution.

EtBr - CsCl density gradient centrifugation
 CsCl density gradient is prepared.  DNA has a buoyant density of 1.7 g/cm3.  EtBr binds to DNA by intercalating between

adjacent base pairs.
  

Results in decrease in buoyant density. Linear DNA – decrease of 0.125 g/cm3. Supercoiled DNA – decrease of 0.085 g/cm3.

 Supercoiled DNA forms a different layer during

centrifugation

Preparation of phage DNA
 Extracellular medium of infected bacterium used

instead of cell extract.  The phage culture is induced to undergo a lytic phase to get a high titre phage concentration in the extra cellular medium.

Visualizing DNA molecules in a gel
 DNA carry a net negative charge – move

towards the positive pole.  Can be separated on the basis of size on agarose gel.  Visualized by staining or autoradiography.  Distance moved by molecule D = a-b (logM)
 

Where a and b are constants depending on electrophoretic conditions, M is the molecular mass

Manipulation of purified DNA
 Manipulation includes
  

Cutting and joining DNA molecules, Shortening and lengthening of DNA molecules, Addition or removal of specific chemical groups. Gene cloning, DNA biochemistry Study of gene structure Study of gene expression

 Provides foundation for
   

 Carried out using specific enzymes.

DNA manipulative enzymes
 Nucleases  Ligases  Polymerases  Modifying enzymes  Topoisomerases

Nucleases
 Degrade nucleic acid molecules by breaking the

phosphodiester bonds .  Two types

Exonucleases – removes nucleotides one at a time from the end of the nucleic acid molecule. Endonucleases – break internal phosphodiester bonds within a nucleic acid molecule.

 Restriction endonucleases cleave at specific sites  Three types – type I, type II and type III

Restriction digestion
 Type II enzymes used.  Recognize specific recognition sequences and

cuts DNA at these sequences.  Some produce blunt ends, while others yield sticky ends.  Have specific requirements of temperature and Mg2+ concentration.  Results in Star Activity if requirements are not met properly.

Restriction mapping
 Relative positions of recognition sites of a number of

restriction enzymes on a DNA molecule.  A series of restriction digestion performed on DNA and fragments produced by each enzyme are determined by electrophoresis.  The fragments are then compared with size markers.  This information is supplemented by a series of double digestions.

Ligases
 Two phosphodiester bonds, one for each strand

is made to link two DNA molecules.  Both blunt and sticky ends can be ligated.  Sticky ends are ligated more efficiently.  Adaptors, Linkers and homopolymer tailing are used to generate sticky ends.

Polymerases
 Synthesize a new strand of DNA complimentary

to an existing DNA or RNA template.  Requires a primer.  DNA pol I has duel activity
 

DNA polymerization DNA degradation (Nick translation)

 Klenow fragment – fragment of pol I retains the

polymerase activity but lacks nuclease activity.

DNA modifying enzymes
 Alkaline phosphatase – from E. coli or calf

intestinal tissue – removes phosphate group from 5’ terminus.  Poly nucleotide kinase – from E. coli infected with T4 phage – adds phosphate groups to free 5’ terminus.  Terminal deoxy-nucleotidyl transferase – from calf thymus tissue – adds one or more deoxynucleotides on to the 3’ terminus.

Topoisomerases
 Change the conformation of covalently close-

circular DNA by introducing or removing supercoils.  Very important in replication.  Use in genetic engineering debated.

Transformation – the uptake of DNA by bacterial cells
 Uptake and stable retention of plasmid detected

by expression of genes carried by plasmid.  Most species uptake DNA from media naturally – particularly if the DNA molecule is a plasmid with an origin of replication recognized by the host.  Competent cells have higher transformation efficiency.

Preparation of competent E. coli cells
 Cells soaked in ice cold 50 mM CaCl2 solution.  CaCl2 precipitates DNA on cell surface.  Actual uptake facilitated by heat shock at 42 0C.  Heat shock produces transient pores on the cell

membrane.

Other methods for transformation
 Transfection – recombinant phage DNA

   

packaged into phage heads and allowed to infect cells. Protoplasts and Electroporation. Microinjection. Gene gun. Viral infection.

The problem of selection
 Ligation mixture contains  Desired recombinant molecule.  Unligated vector molecule.  Unligated DNA fragments.  Vector molecules that have re-circularized, without new DNA being inserted (self ligated vector).  Recombinant DNA molecules that carry the wrong inserted DNA fragment.

Success or failure of gene cloning studies depends on whether or not a strategy can be devised by which clones of the desired gene can be selected directly, or alternatively, distinguished from other recombinants. Once this problem is resolved, and a clone has been obtained, a wide variety of information can be extracted from the gene

Strategies for obtaining the correct clone.
 Direct selection for the desired gene.  Identification of clone from a gene library.

Direct Selection
 Selectable markers carried by recombinant

DNA.  Antibiotic resistance, specific enzyme activity etc used as markers.  Insertional inactivation of selectable markers is another approach.  Insertional inactivation detected by replica plating.

5-bromo, 4-chloro 3-indodyl – β D galactopyranoside + isopropyl thiogalactoside

Direct selection by marker rescue
 Genes that code for biosynthetic enzymes are

used.  A mutant host strain which lacks the specific enzymatic activity is used (auxotrophic).  The correct recombinant construct contains the gene coding for the enzyme.  Only those hosts that contain the correct DNA construct (non – auxotrophic) can survive on a minimal media.

Identification of gene from a gene library
 A genomic library is a collection of clone

sufficient in number to be likely to contain every single gene present in a particular organism.  Total cell DNA is purified and partial restriction digestion is carried out.  Resulting fragments are cloned into suitable vectors.  cDNA libraries are much easier to handle.

c DNA libraries
 Not all genes are expressed at a particular time.  Total mRNA copy of a cell at a time represents

the genes expressed.  mRNA can be reverse transcribed to give cDNA.  The resulting cDNA clones will represent the mRNA present in the original preparation.  A library of cDNA will be much smaller than a genomic library

Any two single-stranded nucleic acid molecules have the potential to form base pairs with one another. With most pairs of molecules the resulting hybrid structures are unstable, as only a small number of individual inter-strand bonds are formed. However, if poly-nucleotides are complementary then extensive base-pairing will occur, to form a stable double stranded molecule. This can occur between ss DNA , ss RNA & also between a ss DNA and an ss RNA

Colony & Plaque hybridization
 Colony or plaque transferred to nitro-cellulose     

membrane. Membrane treated to remove all contaminating materials. DNA alone remains on membrane. DNA denatured to produce ss DNA molecules. ss DNA bound to nitro-cellulose membrane by raising temperature to 80 0C. DNA binds to membrane through the sugar-phosphate backbone, bases remain free to base pair.

Colony & Plaque hybridization…
 Labeled probes added to membrane.  Probes base pair with complementary regions.  Labels can be radioactive, fluorescent (biotin,

avidin,) or chemi-luminescent (HRP, luminol).

Applications
 Abundancy probing to analyze a cDNA library.  Oligonucleotide probes for genes whose

translation products have been characterized.  Heterologous probing to identify related genes.

Locating the position of a cloned gene on a DNA molecule
 Restriction digest probed with labeled probe.  Can be done when restriction fragments are still

on the gel.  This is cumbersome and yields false positive results.  Hence restriction digest is transferred on to a nitro-cellulose membrane – Southern transfer

Southern Blotting
 Perfected by Prof. E M Southern.  DNA bands from gel transferred on to

membrane, using buffers.  Same method can be used to transfer RNA (Northern) and Protein (Western) molecules.  Advanced methods couple electrophoresis with blotting – electro blotting.

Separating large DNA molecules by electrophoresis
 Orthogonal Field Alteration Gel Electrophoresis

(OFAGE).  Contour Clamped Homogeneous Electric Field (CHEF).  Field Inversion Gel Electrophoresis (FIGE).

in situ Hybridization
 Provides a direct visual of a cloned gene on the

light microscopic image of a chromosome.  Cells treated with a fixative to a glass slide.  Treated with ribonuclease and NaOH to degrade RNA and denature DNA.  Base pairing between individual polynucleotide strands is broken and chromosomes unpack to a certain extent.

in situ Hybridization…
 A sample of cloned gene is then labeled and

applied to the chromosome preparation.  Hybridization occurs and a dark spot results after autoradiography.  If a fluorescent marker is used the technique is called FISH (Fluorescent in situ hybridization)

Chromosome walking
 When the position of the gene is known, but no    

probe is available. A probe for a near by gene is used. Two clone libraries prepared with different restriction enzymes required. The clone containing the known gene from library A is used to hybridize library B. One of the positive hits in library B is used to hybridize library A.

Chromosome walking…
 This process is continued, gradually building up

a partial restriction map of overlapping fragments.  Tedious technique.  Longest walk ever carried out till date is only 250 kb in length.

Restriction Fragment Length Polymorphism (RFLP)
 The coexistence of multiple alleles at a locus is

called genetic polymorphism. Any site at which multiple alleles exist as stable components of the population is by definition polymorphic.  A change in a single nucleotide when alleles are compared is called a single nucleotide polymorphism (SNP).

RFLP…
 A difference in restriction maps between two

individuals is called RFLP.  Basically a RFLP is a SNP that is located in the target site for a restriction enzyme.  It can be used as a genetic marker in exactly the same way as any other marker.

Genetic fingerprinting
 Specialized form of RFLP.  The mini satellite DNA is probed.  This differs in length in different individuals.  Length of repeats ranges from 9 to 40 bp.  Number of mini satellite repeats ranges from 10

to 30.  Variability due to loss or gain during replication.

Genetic fingerprinting…
 A mini satellite pattern (fingerprint) represents

the repertoire of the lengths of some of these sequences in an individual.  The chance of finding two individuals with the same pattern is about one in 105 – 108.

Electro Motility Shift Assay (EMSA)
 Used to identify DNA-protein interactions.  Protein bound DNA has relatively lesser mobility

compared to free DNA.  Can be used to determine DNA region binding to protein.  Identifies regulatory elements in genes.

DNA foot printing
 Interaction of regulatory protein protects DNA    

from degradative action of endonucleases. The DNA fragment is end labeled with a radioactive marker. The DNA is mixed with the protein. The mixture is then treated with DNase I. The length of DNA where the protein binds is protected.

Hybrid release translation (HRT) & Hybrid arrest translation (HART)
 Used to identify translation product encoded by a cloned     

gene. cDNA clone is prepares from mRNA. For HRT cDNA is immobilized on nitrocellulose membrane and incubated with mRNA. Specific cDNA gets hybridized and remains bound to membrane. These are now retrieved and translated in cell free system. The protein mixture is run on PAGE

HART…
 For HART, the cDNA is slightly denatured and

added to mRNA sample.  Hybridization occurs, and this hinders translation in a cell free system.  Only non-hybridized cDNA undergoes translation.  So all proteins except that coded by the mRNA is produced.

Polymerase Chain Reaction (PCR)
 Selective amplification of chosen region of a

DNA molecule.  The border sequences of the DNA of interest should be known.  Oligonucleotides complementary to the border sequences act as primers.  Taq polymerase is used.

Primer designing
 Complementary to the sequence flanking the    

gene. The 3’ ends of the hybridized primers should point towards one another. The gene should not be greater than 3kb and ideally be less than 1kb. Length of primer should be considered. GC content should be high.

Correct temperatures to be used
 Denaturation temperature – ~94 0C.  Annealing / Renaturation temperature – ~ 45 –

55 0C.  Extension / Synthesis temperature – ~75 0C.  Annealing temperature should be less than Tm. Tm = (4 x [G + C]) + (2 x [A + T]) 0C

Types of PCR
 Gene Synthesis PCR  Overlapping primers used to produce desired DNA.  Reverse Transcriptase PCR (RT – PCR)

A reverse transcriptase step is included in the first step followed by normal PCR. Yields cDNA from mRNA.

 Real Time PCR (RTPCR)  Tagged primers used to determine concentration on PCR product during reaction.

Random amplification of polymorphic DNA (RAPD)
 Another method of genetic finger printing.  Random primers used to hybridize with total

genomic DNA.  PCR performed to amplify polymorphic DNA.  Each primer produces unique pattern for each individual organism.  Usually used to differentiate between different variety of plant cultivars.

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