Abstract

New creation is possible only BY GOD, (NATURE is

GOD)with ITS/HIS permission, Biotechnology can do the same and

give shape to the new creation and the explored and extracted

miraculous knowledge in Molecular biology has brought a revolution

in constructing a new life for the sake of new and improved life in

the universe. Like modifying organism or plant to improved quality

or inventing a new drug or vaccine or improving the quality of a life

of human or an animal … etc

All the modifications that are carried out in biotechnology is

influenced by the potential knowledge in Molecular biology

experiments and also the changes that are made at the DNA level

which is the material that is a blue print to life dealt with DNA RNA

and Protein which are phenotypic and genotypic expression factors of

all living organism. Studies on DNA sequence is unlimited and every

time, it show its uniqueness, any simple changes affects its bases

(mutation) reflects very seriously in both phenotype & genotype

performance of living things.

1
 DNA sequencing is an important technique which reads exact

bases of DNA present in the given sample. It plays very important

role in all type of Molecular biology applications such as confirmation

of cloned gene sequence, SNP (Single nucleotide polymorphism)

studies, Cancer caused by various type of mutations etc.,

There are more than 1000 samples are sent for Sequence

everyday and Sequencing Companies are grown in dozen in each

states of India. This indicates the importance of DNA sequencing

which needs purified DNA.

The source of DNA for DNA sequencing is from PCR

amplification, restricted fragments, cloned Plasmid, etc., Among all

other source, the most frequent sample received by DNA sequence

company is PCR sample.

There are somany methods available now a days to recover

PCR amplified products from the Agarose gels. The question now

arising is how much purity is there in isolated DNA or RNA and

PCR amplified product ? Don’t we increase the purity of the PCR

products rapidly with less expensive?

2
 The present work was aimed in obtaining highly purified DNA

sample so that sequence results will not interfered with at find a best

method to suggest for our scientists that must be easy and

reproducible. Most commonly used purification protocols are

selected and tested on the basis of

• Efficiency of purification.

• Time taken to perform purification of PCR products

• Chemical expenditure.

We followed different available protocols for recovering of

pure PCR products and we selected four good protocols among

the available methods and with small modifications we achieved

our target and fulfilled our thoughts in our entitled “Simple &

Reliable method to purify PCR amplified DNA sample for DNA

sequencing “ protocol and we feel this is the best protocol.

3
 Introduction

Cell is the structural and functional units of every living

organism. Growth is the common function of every living

organism,shown through the multiplication of cell number.For each

and every cell division the daughter cells should share all the

characters of their parents.The parental charecters are transferred to

their daughters through some factors,because of this the same type of

the organisms have been existing in universe for millions of the

years from the life has began on earth.That factor is nothing but

genes, the total set of genes in an organism is called genome.

The genetic material is of two types.

1) Deoxy ribo nucleic acid (DNA) and

2) Ribo nucleic acid (RNA).

4
 Molecular biology

All the DNA based experiments are come under Molecular

biology Molecular biology is a well developed branch of
Biotechnology.Though the Carbohydrate, Proteins and lipids are
essential biomolecules ,Molecular biology donot concentrate on
them ,It deals with the structural and functional information of the
genetic material because the Carbohydrate, Proteins and lipids
biosynthesis information is kept in genetic matarial (DNA) and they
are processed from the DNA through various cellular mechanisms.

Central dogma of Molecular Biology

The life of an organism starts from a single cell stage,generally

called zygote.The result of multiple divisions of the zygote form the

massive and well organized body give life and shape to the

organism.Body of the organisms are composed of the above

mentioned biomolecules called carbohydrates,proteins and lipids.

All the information of synthesis of the Biomolecules is well

furnished in the genetic material and by the catabolic mechanisms,

they are synthesized .

5
 These mechanisms are
1)Replication(Mechanism of duplication of DNA),
2)Transcription(Synthesis of mRNA from DNA ) and

3)Translation (Synthesis of Proteins from mRNA).

DNA

↓ Replication

DNA

↓Transcription

RNA

↓Translation

PROTEIN

Central dogma of Molecular Biology

Expression of every character of organism is nothing but gene

expression which is nothing but protein synthesis from the respective

gene.Molecular biology dealt with modification of genome

6
expressions and gene maipulations so that wished characters can be

bring out from the Genetically Modified Organisms

(GMO).Biotechnology has achieved a lot in the mean while short time

,it has grown very fast with in the 50 years.

Molecular biology has reached a stage of making a man long

life from the first step discovered genetic material.It can make a man

disease free,It can findout the genome expression forecast.It can

mould a life according to our wish.

Achievements in Molecular biology

We can imagine how much Molecular biology based research

has been going on with single evidence that nearly 60 nobel prizes

were given for various Molecular works since 1958.

Molecular biology has enterd into all the fields like

agriculture,food and technology, dairy, Pharmacy, Immunology,

Fermentation, stem cell biology,entomolgy,oncology etc… and every

day it is invening somany great things.

7
  Molecular biology has given a new life to the victims of

dreadful diseases like Parkinson’s disease,Alzheimer’s disease etc by

transplaning healthy nerve cells into their brains.

 It created Genetically Modified Plants and animals with

high productivity and with high resistance towards harmful diseases.

 Ian wilmat Like scientists in 1997 created a sheep Dolly

by using Cloning technique was a part and parcel of Molecular

biology.

 Artificial Insuline production is a great relief to the

diabetic patients is another great help done by Molecular biolgy for

the human because every year nearly 10 millions of the sugar patients

al over the world have been saving their lives by consuming

Insuline.

 It has established the whole genome information in

Human under Human genome project in the year 2000,a great mile

stone in the history of Biotechnology.

 Genetically engeneered micro organisms have invented

with the knowledge of recombinant DNA technology (r DNA

techonology).

8
 In this way the fruits of Biotechynology and Molecular biology

research have wide range of applications.infact there is no other

branch of science which has as many applications as

biotechnology.Biotechnology has benefited medical and health

sciences(diagnostics, therapeutics,foods), agriculture science

(improved crop yield ,food quality , improved animal health) and

environmental sciences (pollution control,environmental

monitoring ,bioremidiation).

DNA sequencing

Since all the molecular biology work is linked with the DNA

and its separation from the cell,cutting the gene of interest and

inserting in the wanted genome etc.. .,the separated DNA should have

the gene of interst with out any error of nucleotide base sequance.we

can attain a new gene expression only when inserted the particular

gene of interest with out ant nucleotide errors in the host genome.

For that before inserting the gene we should sure of its purity

and it can be declared by DNA sequencing.

9
 DNA sequencing provide all the information of the sequance

of the given gene so that it decides whether to proceed or not to

select that isolated gene.

There are more than 1000 protocols are available for the

isolation and purification of the genes and PCR products,but all are

not that much applicable all times.Some are costly ,some are time

consuming and some are not giving 100% results. We focused on the

best available methods and with little modifications,we achieved a

riliable, rapid and low expensive method for isolation and

purification of PCR products.

10
 Review of literature

Before going to work with any molecular tool the first and foremost

important thing is Isolation of DNA by various methods. Best

methodology to study the sequence of genome of an organism is

selection of smaller genome.Phage lambda DNA is an example for

such investigations.Coli phage lambda DNA is a widely used vector

for recombinant DNA. The middle third of its 48,000 bp contains no

genes required for lytic growth and is, therefore, replaceable. The

usual recombinant lambda DNA contains 80% lambda vector DNA

and 20% insert, as compared to a usual cosmid DNA that contains

10% vector and 90% insert.

Agarose gel electrophoresis

Agarose gel electrophoresis is a method used in biochemistry

and molecular biology to separate DNA, or RNA molecules by size.

This is achieved by moving negatively charged nucleic acid molecules

through an agarose matrix with an electric field (electrophoresis).

Shorter molecules move faster and migrate farther than longer ones

( Sambrook J,et al., 2001). It is usually performed for analytical

11
purposes, but may be used as a preparative technique prior to use of

other methods such as mass spectrometry, RFLP, PCR, cloning, DNA

sequencing, or Southern blotting for further characterization (Berg

JM, et al.,2002).

The electrophoresed DNA was observed under Uv light for

visualizing bands. The main source for visualisation of DNA under

electrophoresis is Ethidium bromide which acts as intercalating agent.

Ethidium bromide binds to the base of Nucleotides and emits the

orange colored fluorescence when observed under UV light ( Milan

Bier,1959).

Improvements in electrophoresis technology have been

announced recently by various companies (Molecular Dynamics,

Perkin-Elmer/ABI, Beckman Coulter). In particular, the 96-sample

capillary electrophoresis instruments being released promise increased

throughput and decreased cost for DNA sequencing. Despite improved

separation time, automation, and sample tracking compared to slab

gels, capillary technologies have not gained acceptance in the past

because of difficulties in resolving long DNA strands.

12
 Recent developments in sequencing reaction purification Ruiz-

Martine et al. 1998 (Ruiz-Martinez, M.C., et al.,1998) and their

effect on read length have made it more likely that capillary

electrophoresis technologies will supplant slab gel-based sequencing

instruments in the near future.

Consequently, there is an increasing demand for high-

throughput, low-cost methods for the preparation of samples to supply

these instruments. Commercially available methods for high-

throughput sample purification (Qiagen) are expensive (~$1 per

sample for the purification kit only; we estimate the total cost of

sample preparation for most centers to be between $1.50-3.00). The

more common centrifugation-based protocols are very labor intensive.

The high sample-preparation costs for sequencing centers that rely on

these methods will offset savings resulting from advances in

electrophoresis systems.

Submarine AgaroseGel Electrophoresis Instrument

13
Amplification of gene of interest by Polymerase
chain reaction.

The polymerase chain reaction (PCR) is a technique widely

used in molecular biology. It derives its name from one of its key

components, a DNA polymerase used to amplify a piece of DNA by

in vitro enzymatic replication. As PCR progresses, the DNA thus

generated is itself used as template for replication. This sets in motion

a chain reaction in which the DNA template is exponentially

amplified. With PCR it is possible to amplify a single or few copies of

a piece of DNA across several orders of magnitude, generating

millions or more copies of the DNA piece. PCR can be performed

without restrictions on the form of DNA, and it can be extensively

modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA

polymerase, such as Taq polymerase, an enzyme originally isolated

from the bacterium Thermus aquaticus. This DNA polymerase

enzymatic ally assembles a new DNA strand from DNA building

blocks, the nucleotides, using single-stranded DNA as template and

DNA oligonucleotides (also called DNA primers) required for

14
initiation of DNA synthesis. The vast majority of PCR methods use

thermal cycling, i.e., alternately heating and cooling the PCR sample

to a defined series of temperature steps. These thermal cycling steps

are necessary to physically separate the strands (at very high

temperatures) in a DNA double helix (DNA melting) used as template

during DNA synthesis (at lower temperatures) by the DNA

polymerase to selectively amplify the target DNA. The power and

selectivity of PCR are primarily due to selecting primers that are

highly complementary to the DNA region targeted for amplification,

and to the thermal cycling conditions used.

Developed in 1983 by Kary Mullis, PCR is now a common and

often indispensable technique used in medical and biological research

labs for a variety of applications. These include DNA cloning for

sequencing, DNA-based phylogeny, or functional analysis of genes;

the diagnosis of hereditary diseases; the identification of genetic

fingerprints (used in forensics and paternity testing); and the detection

and diagnosis of infectious diseases. In 1993 Mullis won the Nobel

Prize for his work on PCR.

15
 A common requirement in the manipulation of nucleic acids to

perform sequencing, cloning or hybridization assays is polymerase

chain reaction (PCR) amplification of a gene ( a DNA region) of

interest. The result of this reaction is the amplified desired sequence

along with a mixture of nucleotides, primer oligonucleotides, and

buffer components. This sequence of interest is usually purified prior

to subsequent manipulation. Many methods are employed in this

effort including gel electrophoresis, silica based affinity matrices,

precipitation, and ion exchange or size exclusion chromatography

columns. For low through put applications, these matrices are applied

to single spin column devices.

The polymerase chain reaction (PCR) has only recently become

a powerful tool for the detection of microbial organisms from

environmental samples. The application of this molecular technology

has so far been hampered by the presence of various impurities co-

extracted with soil DNA, such as humic acids and metal ions. These

interfering substances can inhibit PCR most likely due either to

chelation of humic acids with magnesium ions required by Taq

polymerase, or to the binding of primers that reduce the sensitivity of

detection (Milan Bier, 1959). A number of works devoted to

16
methodological approaches outline the difficulty to recover pure DNA

extracted from soil or sediment samples. Removing humic substances

from the DNA samples represents actually a methodological

challenge. Time consuming methods like CsCl gradients (sai, et al.,

1992,Ogram , et al., 1987) or extensive and repetitive precipitation

steps (Holben, 1988) have been developed. Methods such as diluting

crude extract samples have been used but this latter reduces the

detection limit (Milan Bier, 1959). In order to simplify the purification

steps, the use of various minicolumns has been recently tested by

some authors (Tsai,et al., 1992, Porteous, et al.,1997, Zhou, J ,et

al.,1996, Purday,et al.,1996, Tebbe,et al.,1993).

Impartance of purification of PCR product.

PCR products must be purified before sending DNA

sequencing. If there is small contamination of primer, template DNA,

etc will leads to nonspecific sequencing or unclear data sequencing, so

that it will be useless to obtain.

Main source of contamination in PCR products is template

DNA and Primer mix.

17
 To remove both from PCR products, currently many scientists

are using gel extractions methods and even conventional methods of

precipitation using phenol.

A rapid and simple method for the recovery of DNA fragments

from an agarose gel are very essential for molecular cloning

processes.It involves a ten minute centrifugation of the DNA-

containing gel layered on a GeneScreen (NEN) or a Durapore

(Millipore, GVWP 04700) membrane. With the latter a recovery of

about 70 percent has been obtained with DNA fragments in the range

of 0.4 to 25 Kbp. The eluted DNA stays intact and has been used

successfully for ligation, restriction digestion, fill-in reaction by

Klenow polymerase, tailing and for priming reverse transcription.

Therefore, it is particularly useful for construction of plasmids and for

other recombinant DNA research (Aaij, C,et al.,1972). The excellent

commercially extracting DNA kits are available by so many

manufacturers include Qiagen, Sigma, Novagen if you afford them.

They cost 1–2 US$ each.

18
 The purification of DNA fragments from the agarose matrix for

further study has long been problematic. Although several techniques

have been devised for this purpose (Sharp, P. A., et al.,1973, Allet, B.,

et al.,1973), none has satisfactorily eliminated the problems of

incomplete separation of agarose from DNA, degradation or other

modifications of DNA, low yield, inconvenience, etc. (Roberts, R. J. ,

1976). The problems are often magnified when large preparative gels

are used or when multiple samples are analyzed.

DNA sequencing

DNA sequencing is a fundamental process in the biological

sciences. In 1977, Sanger's group reported an enzymatic method for

DNA sequencing (F. Sanger, et al.,1977). They generated a nested set

of radioactively labeled DNA fragments in four reactions, one for

each terminal dideoxynucleotide. The reaction products are separated

by size in adjacent lanes of a high resolution polyacrylamide gel and

detected by use of autoradiography. The sequence is interpreted from

the pattern of alternating bands in the lanes corresponding to the

terminal base of the fragment. Sequence determination by this classic

technique is an important, albeit tedious and labor intensive, task. An

19
advance in sequencing technology occurred in 1986-88 when Smith

and co-workers in Hood's laboratory, workers in Ansorge's group,

Prober and co-workers at DuPont, and Kambara and coworkers at

Hitachi reported DNA sequencers that replaced the radioactive labels

and autoradiography in Sanger's method with fluorescent labels and

laser-based detection ( L.M. Smith, et al.,1986, W. Ansorge, et

al.,1986, J.M. Prober,et al.,1987, H. Kambara, et al.,1988).

The enzymatic chain termination sequencing method developed

by Sanger et al.( Sanger,F,et al., 1977)J has become, together with the

chemical sequencing method of Maxam and Gilbert (Maxam, A.M.,et

al., 1980) a key tool in molecular biology, especially in combination

with M13 phage vectors as a source of single stranded DNA

sequencing templates (Messing, J.,1983). As more demanding

sequencing projects are being contemplated (Dulbecco, R.,1986), and

with the development of fast automated DNA sequencing systems

(Smith, L.M., et al.,1986, Ansorge, W., et al.,1986, Ansorge, W., et

al.,1987), the method presently used for preparation of DNA

templates may prove to be one of the limiting factors for the

acquisition of new sequence information. The standard methods are

20
reliable but slow, and not easily amendable for automation, involving

purification of the phage by precipitation with polyethylene glycol,

followed by deproteinization by phenol and chloroform extractions

and concentration by ethanol precipitation of the DNA.

At Stanford, a modular, integrated, and automated system is

being developed for shotgun sequencing DNA at a rate of

10,000 sequencing lanes per day, and at a cost of $0.70 per lane. In

June of 1997, the first half of this system, here referred to as the

"Front End," was completed. This subsystem produces sequence-

ready templates from dishes of M13 plaques at a throughput and cost

compatible with the stated goals, and is therefore ideal for use with

new, high-throughput electrophoresis technology. The total cost of

plating, picking, growth, and template purification with this system is

$0.29 per sample, 5-10 times less than the cost of the equivalent

processes employed at many sequencing centers. Though other

integrated, automated systems Hawkins et al. 1997 (Hawkins,et

al.,1992) have been constructed for template purification and cycle

sequencing, no other proven automated systems exist that are capable

21
of producing templates from dishes of microbial clones at this

throughput and cost.

DNA sequencing has emerged as a leading

diagnostic method in clinical laboratories (Bentley D R.,2004, Bell

J.,2004). The original procedure based on the use of dideoxy

terminators (Sanger F, et al.,1977) has undergone many

improvements, including the development of alternative approaches

(e.g., pyro sequencing and sequencing by hybridization), making DNA

sequencing feasible and indispensable in molecular diagnosis of

hereditary disorders, detection of somatic mutations, and tracing of

single-nucleotide polymorphisms. One widely used strategy for DNA

sequencing is based on analysis of large DNA molecules by primer

walking (Strauss EC, et al., 1986). This strategy makes use of

individual primers specifically designed for each sequencing step and

enables direct and systematic analysis of DNA fragments on both

strands with low redundancy and without subcloning. The primer-

walking strategy is attractive for large-scale projects, but primers are

expensive and delays are associated with their design and synthesis.

22
 Materials

ISOLATION OF DNA LAMBDA PHAGES BY

PHENOL-CHLOROFORM METHOD

Materials

E. coli C600,

LB Agar medium

TE buffer( 0.01 M Tris, and 0.001 M EDTA, pH 8.0; diluted from a

100x stock: 1.0 M Tris, 0.1 M EDTA, pH 8.0.) , 0.5 M EDTA( pH

8.0),:

10% SDS: (10 g of sodium dodecyl sulfate dissolved in 100 ml

waterm Ethyl alcohol: 95 or 100% and 70%, both at -20°C.)

Phenol: (TE buffer-saturated phenol. Added the buffer to the phenol

and shaken and poured in an amber bottle. ,Allowed to stand

overnight in a refrigerator,Chloroform ,

23
Phenol: Chloroform (1:1): (Mixed one volume of buffer-saturated

phenol with the same volume of Chloroform in an amber bottle.

Shaken and placed in the refrigerator overnight. For this procedure,

the following reagents are required:

0.1 M MgSO4,

DNase I (free of RNase, 100 µg/ml in 0.01 M Tris, pH 8.0)

RNAse (free of DNAse).

Specialized reagents for growing coli phage lambda:

20%Maltose ,

SM buffer (NaCl 5.8 g ,MgSO4·7H2O 2 g , and 50 ml 1 M Tris pH

7.5 mixed in 1 Liter water)

Gelatin (2%).

24
ISOLATION OF LAMBDA PHAGE DNA BY
COLUMN METHOD
Materials

Lysate

DNAgeI

1mg/ml DNAseI stock solution in sterile 150 mM NaCl 50% glycerol

was Prepared and stored at -20 °C.

3M KOAc

29.4 g of potassium acetate in 100 ml of distilled water was dissolved

and autoclaved and stored at room temperature.

Proteinase K (20 mg/ml stock)

20 mg of proteinase K was dissolved in 1ml of sterile distilled water

and stored at -0 °C . Before using, made a 1:200 dilution in distilled

water to give a 0.1 mg/ml dilution.

Muscle glycogen (10 mg/ml stock)

10 mg of muscle glycogen was dissolved in1ml of distilled water,

filtered, sterilize and stored at 4. Before use make a 1:10 dilution in

sterile dH2O to give a 1 mg/ml dilution and store at 4°C.

25
DEAE-cellulose

DE52 (Whatman preswollen) was prepared in the hydrogenated form

as follows:

In a 2L flask, 500 g of DE52 was suspended in 1.5L of 0.1N HCl.

After the DE52 had settled decanted supernatent and DE52 was

stored at 4 °C as 1:1 slurry in 10 mM Tris-HCl pH8 and mixed the

slurry well and brought to room temperature before using.

Special supplies required

BioRad Econo Columns and

Whatman DE-52 .

Agarose gel electrophoresis

Meterials

Agarose powder,

TAE buffer (Tris acetate EDTA) ( 10mM Tris-HCl (pH-7.4), 1mM

EDTA (pH-8.0) (stored at room temp.)

Ethedium bromide,

10% SDS( 10gm of SDS was dissolved in 100 ml of Distilled water)

26
Agarose gel electrophoresis kit, purchased from Helini Biomolecules ,

Chennai. DNA amplification procedure

Chemicals & Reagents

Lambda DNA,

PCR MasterMix,

Lambda

RNase: 10mg/ml purchased from HELINI Biomolecules, Chennai.

Proteinase K 10mg/ml purchased from HELINI Biomolecules,

Chennai. Spin-columns (Nucleic acid purification columns)

method

Materials:

6M Guanidium Chloride,

70% Ethanol

PCR purification kit purchased from HELINI Biomolecules, Chennai.

27
Method 4

Enzymatic purification

Materials

PCR mixture (directly after PCR) 5µl

Exonuclease I 0.5µl

Calf intestine alkaline phosphatase 1µl

Method 1

Purification of PCR Products using Phenol mix extraction

Materials:

Phenol/ Chloroform (1:1) Equal volume of Phenol, Chloroform was

mixed.

5M sodium Chloride

95% Ethanol: 95 ml of ethanol was mixed with 5ml of distilled water.

28
5M ammonium Acetate

Isopropanol

70 % ethanol: 70 ml of ethanol was mixed with 30 ml of distilled

water

Silica gel method

Materials

Silica,

PBS,

Sodium iodide,

29
 Methodology

ISOLATION OF DNA LAMBDA PHAGES BY

PHENOL-CHLOROFORM METHOD

Methods

Preparation of fresh E. coli colony

0.5ml E.Coli was picked into 5 ml of LB Agar medium and mixed

with 0.2% maltose and placed on a wheel at 37°C for

approximately 4 hr.

In sterile conditions, the culture was transferred to a sterile 15-ml

conical tube and the E. coli were pelleted by centrifugation in

an SS34 rotor with adaptors in an RC-5B centrifuge for 8 min

at 4 K rpm.

The medium was poured off gently. The bacterial pellet was

resuspended in 4-6 ml of 0.01 M MgSO4 by pipetting up and

down and E. coli culture was prepared.

prepared E. coli culture was incubated at room temperature for 4 days.

30
 Isolation of DNA from Phage lambda

10 µl of E. coli culture and 0.2 ml SM buffer (no gelatin) were

mixed in a small tube.

Phages were added to the E. coli and SM buffer, and the tubes were

placed on the 37°C wheel for 20-30 min.

By using the Pasteur pipettes, picked up the plaque and expelled into a

sterile eppendorf tube by a wrist flick.

0.5 ml of SM buffer (no gelatin) and 2 drops of chloroform were

added to the plaques.

The eppendorf tube was allowed to stand for 2-4 hr at room

temperature overnight to elute the phage.

A phage stock was made from the isolated plaque. 5-20 µl of phage

was added to the mixture of LB Agar, SM buffer and 10µl of E.

coli in small tube and placed on a 37°C wheel for 20-30 min.

LB top agar was added to the above tube and vortex for 10 sec.

The above content was poured onto a warm LB agar plate.

After the top agar hardened in 10 min, inverted the plate and placed

at 37°C overnight.

Fully infected, disrupted E. coli lawn was observed on plates.

31
5 ml of sterile SM buffer (no gelatin) was added to that and made the

plate completely covered with buffer.

Gently rotated the plates at room temperature for 1-2 hr and harvested

the buffer using a sterile pipette into a sterile conical 15-ml

tube.

0.2 ml of chloroform was added and vortex for 10 sec.

Spinned the tube in an SS34 rotor with adaptors, RC-5B centrifuge at

4 K rpm for 8 min.

Decant the clear yellow supernatant into a fresh, sterile, 15-ml

conical tube. (debris was removed in this way)

One drop of chloroform was added to the culture and stored at 4°C.

The lambda stocks were diluted ten-fold in SM buffer and gelatin.

LB agar plates were kept at 37°Cand LB top agar was melted, a

liquated, and maintained at 50°C and 0.2 ml of SM buffer and

10 µl of E. coli were taken in small tubes.

Appropriate dilutions were added to the bacteria. The tubes were

placed on the 37°C wheel for 20-30 min.

3 ml of top agar was added to the mixture of phage and bacteria and

Vortex for 10 sec and mixture was Poured onto an agar plate.

32
Rocked the plate by hand to reach an even distribution of top agar

and allowed the top agar to solidify at room temperature for 10

min and Placed the inverted plates at 37°C overnight.

Well-isolated, accurate plaques were observed. Agarose was added to

the plaques to remove the impurities present in agar culture.

The large LB agarose plates were equilibrated at 37°C.

0.2 ml of SM buffer was taken in a small tube and 20 µl of E. coli

culture was added.

lambda was added to the bacteria and SM buffer and placed the tube

on the 37°C wheel for 20-30 min. and vortex for 10 sec, and

pour onto the warm 150-mm plate.

Rotate (rock, swirl) the plate by hand to achieve an even distribution

of top agarose. Allowed the top agarose to harden for 10 min

and inverted the plate at 37°C overnight.

Confluent lysis was observed on the plates and 8 ml of SM buffer

was added to plate and Made the buffer covers the entire plate

The plate was rotated gently at room temperature for 1 to 2 hr. The

phage was eluted into the buffer.

Decanted (pipette) the buffer into a 15-ml conical tube (5-6 ml/plate).

Centrifuged the tube in an SS34 rotor with adaptors, RC-5B

33
 centrifuge for 8 min at 4 K rpm). The debris was pelleted and

most of the phage was reamain in the supernatant. Decant the

supernatant (9-12 ml) to an Oak Ridge tube.

Magnesium ions containing SM buffer and 0.1 ml of DNase I was

added to the above content and placed the tube at 37°C for 2

hs,so that contaminated E.Coli DNA was digested.

0.5 ml of 0.5 M EDTA, (pH 8) and 0.5 ml of 10% SDS was added to

the above content and mixed gently to inactivate DNase I and

disrupt the phage particles.

10 ml of phenol(TE saturated phenol)was added to the content and

vortex for 20 sec to remove proteins and SDS and Centrifuged

in an SS34 rotor, RC-5B centrifuge for 10 min at 10 K rpm at

10°C.

Decant 10 ml of the upper, aqueous phase to a fresh Oak Ridge tube

Double the volume of cold (95-100%) ethanol was added to the

above content to increase the concentration of DNA and to

remove traces of phenol and chloroform and Vortex for 10 sec

to mix and Centrifuged in an SS34 rotor, RC-5B centrifuge (or

equivalent), for at least 30 min at 12 K rpm at 10°C. Gently

decant the supernatant.

34
10 ml of cold 70% ethanol was added to the supernatant To remove

ex cess salt,. (should not disturb the nucleic acid

precipitate)and Centrifuged for 10 min at 12 K rpm at 10°C.

The supernatant was gently decanted and 10 ml of cold (95-100%)

ethanol was also added. Centrifuged for 10 min utes at 12 K

rpm at 10°C. and again the the supernatant was decanted. Drain

briefly and air dry briefly and RNA precipitation was observed.

The precipitate was Dissolved in 2.5 ml of TE buffer for 1 hour

0.1 ml RNase (1 mg/ml, DNase free)was added to the above content

and and mixed gently,so that the contaminating RNA was

removed by digestion.

The content was Placed at 37°C for 1-2 hr and 0.1 ml of proteinase K

(1 mg/ml, nuclease-free) was added and incubated at 37°C for

2 hrs so that Contaminating protein, including RNase and

proteinase K itself, was re moved by digestion. And Cooled the

components to room temperature

2.5 ml of phenol:chloroform was added to the above contents and

Vortex hard for 10 sec and Centrifuged for 10 min at 10 K

rpm, 10°C, in an SS34, RC-5B centrifuge

35
 The upper, aqueous layer was decanted to a Centricon and

centrifuged at 5 K rpm in an SS34, RC-5B centrifuge at 10°C

and got the Phage lambda DNA.

ISOLATION OF LAMBDA PHAGE DNA BY

COLUMN METHOD
Mini-prep method for lambda phage DNA purification
from lysates.

Method

1. Lysate was taken in centrifuge tubes and Spined the tubes at 2500

rpm for 30 minutes at room temperature in the Beckman J-6

centrifuge to pellet any cell debris.

Prepared the DEAE-cellulose columns as follows

36
2. The DE-52 slurry was Warmed up to room temperature and

Placed the Biorad Econocolumns in the 2-row column rack and

poured approximately 4 ml of the DE-52 slurry into the column.

3. The height of the packed column was maintained 1.8 to 2 ml and

the excess buffer was flew through (about the halfway point of the

angled portion of the column)

4. The diluted lysate was poured into the column bed.

5. 5 ml of Chase buffer {10 mM Tris, pH8.0, 10 mM Mg(OAc)2, 50

mM NaOAc} was added when the column flow has stopped and

let it was flew through the column.

6. 1 ml of Elution buffer {10 mM Tris, pH8.0, 50 mM

Mg(OAc)2}was added and let it was flew through the column

7. 0.6 ml of Elution buffer was added and collect the flow-through in

sterile eppendorf tubes placed in a Sarstedt rack.

8. Transfered a 10 ul aliquot into another eppendorf tube and stored

it at 4°C.

9. The phage fraction (containing DNA) from the column was

collected and lysed any remaining phage by washed the column

with 10 ml 1N NaOH.

Phage Lysis and DNA Concentration

37
10. 10 ul of 0.1 mg/ml proteinase K (stock solution is 20 mg/ml stored

at -20°C) and 25 ul of 10% SDS was added to the phage fraction

from the column and Mixed and kept at room temperature for 5

minutes.

11. 100ul of 3M KOAc (forms a white cloudy precipitate) was added

to the above content and Mixed and heated at 65°C to 88 °C in a

waterbath for 25 minutes. The precipitate was dissolved.

12.The tubes were cooled in an ice-water slurry for at least 20

minutes. The precipitate again reformed and Spinned the tubes in

an microcentrifuge in the cold room at 12000 rpm for 10 minutes.

13. The supernatant was transferred to a sterile tube containing 20 ul

of 1 mg/ml mussel glycogen

14. 700 ul of cold isopropanol was added to the above and mixed

well and Cool the tubes to -20 °C for 1 hour

15.The tubes in the microfuge were spinned in the cold at 12000 rpm

for 30 minutes and discarded the supernatant.

16.1ml of 95% cold Ethanol was added to the pellet and spinned at

12000 rpm for 5 minutes in the cold room. and discarded

supernatent

38
17.The DNA pellet was airdried with the lid of the tube open at room

temperature for about 10 minutes.

18. The DNA pellet was esuspended in 20 ul of TE (pH8.0) and

vortexed gently with finger and placed the tube in a waterbath at

50°C for 15 minutes to aid resuspension.

. Our motto is to purify the PCR amplified DNA. We

had done our experiments on Lambda phage DNA. Before going to

the amplification the sample were confirmed weather it was DNA or

not. For that we had done electrophoresis and confirmed the eluted

sample was DNA with the orange co lour gel band appeared under

the UV illuminator.

39
 STEP -1

Agarose gel electrophoresis

Method

Preparation of 2% Agarose gel for electrophoresis

1. 2 gms of 2% Agarose was added to 100 ml Buffer provided (50X TAE)

2. Boiled the agarose until it was completely dissolved and made sure no

obvious particle of agarose remain in the suspension

3. When the gel temperature was around 40ºC, 5 ul of ethidium bromide

was added and mixed properly

4. Sealed the gel casting tray on both sides and placed the comb on the

gel tray in appropriate place

5. Poured the agarose mixture into the tray containing comb.

6. After complete solidification of agarose removed the seal from either

sides of the tray without disturbing the gel

7. Kept the gel tray in tank containing 50X TAE buffer with the wells in

the cathode (negative) side. The buffer level in the tank was maintained

above the gel tray.

40
8. Gently lifted the comb without damaging the wells, and the gel was

made ready for loading

9. Connected the power cords between the electrophoresis tank and the

power pack before loading the samples

10. Prepared samples for electrophoresis, by adding 2ul of gel loading dye

into the PCR samples and mixed well by pipe ting

11. 15-20ul of the sample was loaded in 8 wells.

12. After loading, switched on the power pack and adjusted the voltage to

50v or 100v

13. Continued the electrophoresis until the dye reached to 1/3rd of the gel

or above

14. Observed the bands under UV Transilluminator.

41
STEP-2

Amplification DNA sample by PCR

The 8 DNA bands were cut and amplified in PCR.

DNA amplification procedure

Method

1. Taken the PCR tools from -20ºc and keep in ice box.

2. Brefly spined all tubes for few seconds to settle down all the droplets

sticking at the top or sides.

3. Added the following component in to PCR tubes provided

Master mix 24µl

Forward primer 2µl

Reverse Primer 2 µl

Lamda DNA sample 2µl

Distilled water 20µl

Total reaction Mixture 50µl

4. Master mix contain following components,

Distilled water,

Buffer,

42
Taq polymerase

dNTP’s

Total reaction mixture volume is 24 µl.

1. Placed the tubes in thermal cycles and program the number of cycles,

The optimized thermal profile is given below.

Step 1: Initial denaturation at 94ºc for 2 minutes.

Step 2: Denaturation at 94ºc for one minute.

Step 3: Annealing at 60ºc for one minute

Step 4: Extension at 72ºc for 1 minute.

. Step 5: Final Extension at 72ºc for 10 minutes.

Step 7: 10ºc for ever.

Steps 2,3,4 was reapeated for 30 times for the

amplification of desired gene.

2. Collected the tubes from the Thermal Cycler.

43
STEP-3

PURIFICATION OF AMPLIFIED PRODUCT

The amplified PCR products were separated into four sets and each

set containing two amplified PCR products for the purification.

Now in each set of PCR amplified DNA the two samples were

purified in two ways

1) Gel purification and

2) Direct purification.

In a set, one sample was subjected to 2% agarose gel

electrophoresis, and then cut the gel pieces contain DNA bands were

purified through four different selected methods. And the second

sample was directly purified without electrophoressed. The direct

purification method follows the same protocol as gel purification

protocol except gel electrophoresis.

Selected four methods were

1) Phenol mix method,

2) Silica gel method,

3) Spin-column method and

4) Enzymatic method.

44
Method 1

Purification of PCR Products using Phenol mix extraction

Direct purification method

This involved that mixing of 5µl of PCR products with phenol

reagent mix. After brief vortex, three layers were formed where pure

DNA alone stands on the top of the solution and contamination like

Polymerase enzyme, proteins, buffers, chemicals, Mg ions, primers

were trapped in phenol.

Supernatant having DNA was mixed with chloroform to

remove residual effects of phenol and precipitated with iso proponal.

DNA pellet was extensively washed with 70% ethanol to remove any

co precipitated chemicals and finally DNA pellet was dried and

suspended with Distilled water and sent for sequencing in pure tube.

Gel purification method

 Amplified DNA was run on 2% agarose gel.

 Interested DNA fragment was cut and mixed with equal volume of

phenol mix

 Incubated at 65ºC for 10 minutes.

45
 Vortex Intermediately for proper mixing.

 After brief spinning, three layers were formed where pure DNA

alone stands on the top of the solution and contamination like

Polymerase enzyme, proteins, buffers, chemicals, Mg ions, primers

are trapped in phenol.

 Supernatant having DNA is mixed with chloroform to remove

residual effects of phenol and precipitated with iso proponal.

 DNA pellet was extensively washed with 70% ethanol to remove

any co precipitated chemicals and finally DNA pellet was dried

and suspended with Distilled water.

46
Method 2

Silica gel method

Second method for purification of amplified DNA was silica gel

method.

The amplified DNA was subjected to Agarose gel electrophoresis and

obtained the gel piece contain DNA band .

Before purification ,the silica gel has to be prepared.

Silica gel method

Preparation of Silica

1. Suspend 5 g of silica (Sigma, S-5631) in 50 ml of PBS.

2. Allow the silica to settle for 2h.

3. Discard the supernatant containing fine particulate matter.

4. Repeat steps 2 and 3.

5. Spin for 2 min at 2000 g. Discard the supernatant.

47
6. Add 3 M NaI to make a final concentration of 100 mg silica /ml.

7. Store the silica suspension in the dark at 4 C over night.

Recovery of DNA from Agarose Gel Using the Silica Suspension

Direct purification method

 5 ul of 6M NaI was added to the 5 ul of PCR amplified DNA and

mixed with 1 ul NaOAc.

 25 ul of silica suspension was added to the above content and

centrifuged for 10 min.

 The components in the silica precipitation were washed with 500

ul of wash solution.

 The components were air-dried to remove alcohol.

 The silica pellet was resuspended in TE buffer for down stream

digests and heated at 60ºC and spinned in microcentrifuge for 2

min and removed DNA containing supernatant to a cleaned tube

and sent for sequencing.

48
Gel purification.

To the agarose gel containing DNA fragments, add two volumes of 6

M NaI.

Incubate the agarose in 6 M NaI at 55 C for 5-10 min with occasional

mixing.

 Add 10 ul of the silica suspension. Vortex gently. Stand for 5 min

at room temperature with occasional mixing. One mg of the silica

(=10 ul of the silica suspension) binds 3 -4.5 ug of DNA.

 Spin for 1 min in a microcentrifuge. Discard the supernatant.

 Pulse spin, and carefully remove residual liquid.

 Suspend the pellet in 500 ul of 50 mM NaCl, 10 mM TrisHCl pH

7.5, 2.5 mM EDTA, 50%(v/v) ethanol.

 Spin for 1 min. Discard the supernatant.

 Repeat steps 6 and 7.

 Allow the pellet to air-dry for 10 min.

 Add an appropriate volume (at least one pellet volume) of TE.

Vortex gently to resuspend the pellet, and stand for 5 min at room

temperature with periodic agitation.

49
 Spin for 1 min. Transfer the supernatant into a new microfuge

tube and the purified sample was sent for DNA sequencing.

Method 3

Spin-columns (Nucleic acid purification columns) method

Method

Direct purification

After amplification, 5µl of PCR product is loaded in 1% agarose

gelelectrophoresis for confirming amplifications. The rest of the PCR

products is taken for analyses as follows;

 PCR products is directly mixed with 3 volumes of 6M

Guanidium Chloride (Buffer A) and incubated at 50 ºc for 10 minutes

for dissolving the agarose totally.

 After brief spin, sample transferred in to a spin-column and

centrifuged for 1 minute (the DNA remains in the column).

 Washed the column by passing 70% ethanol through (the DNA

remains in the column, salt and impurities are washed out).

50
 Eluted the DNA in a small volume (30µL) of water or buffer,

 Centrifuged at maximum speed to collect the eluted DNA

which send for sequencing.

Gel Purification

 Gel-slice was dissolved in 3 volumes of 6M Guanidium

Chloride (Buffer-A) and incubated at 50 ºc for 10 minutes for

dissolving the agarose totally.

 Applied above mixture into spin-column and centrifuged for 1

minute (the DNA remains in the column).

 Washed the column by passing 70% ethanol through (the DNA

remains in the column, salt and impurities are washed out).

 Eluted the DNA in a small volume (30µL) of water or buffer,

 The eluted DNA was Centrifuged at maximum speed to collect

the DNA pellet and subjected for DNA sequencing.

51
Schematic representation of spin column method

Direct PCR product multiple bands containing

PCR
Product

Loaded in 1% agarose gel

Interest of product cut

Mixed 3 volume of binding buffer

10 min incubate at 55ºC

Transferred to spin column & centrifuge

Maximum speed for 1 min

Column washed with ethanol to remove

Impurities

DNA is eluted from spin column

Send for DNA sequencing

52
 Method 4

Enzymatic purification

Direct purification

 The amplified DNA was directly Taken and all the above

components were Mixed well and incubated at 37ºC for 15

minutes

 Stopped the reactions by heating the mixture at 85ºC for 15minutes

and above product was directly sent for sequencing.

Gel purification

 The amplified PCR DNA product was subjected to 2% agarose gel

electrophoresis and cut the DNA gel piece

 The gel piece was treated with 5µl of PCR mixture (directly after

PCR),

 0.5µl of Exonuclease I and 0.5µl of Calf intestine alkaline

phosphatase and mixed well for 5min and incubated at 37ºC for 15

minutes.

 After well vortex, the reactions in sample were stopped by keeping

the samples at 85ºC

 For 15 minutes and sent for DNA sequencing.

53
 RESULTS & DISCUSSION

DNA sequencing is probably the most important tool available

to the molecular biologist by which the precise order of nucleotides in

a piece of DNA can be determined (Brown, 1990).If the the DNA

sequencing report is satisfactory then we can use the amplified gene

of interest for the further work like cloning ,hybridization,

recombination etc….

We planed to study this to propose a simple and effective

method to purify PCR products.

The main consideration is

1. Purity

2. Effectiveness

3. Time taken (Protocol duration) and

4. cost.

54
We have selected Phage lambda DNA for sequencing throughout our

work because Coli phage lambda DNA is a widely used vector for

recombinant DNA. The middle third of its 48,000 bp contains no

genes required for lytic growth and is, therefore, replaceable. The

usual recombinant lambda DNA contains 80% lambda vector DNA

and 20% insert, as compared to a usual cosmid DNA that contains

10% vector and 90% insert.

Wild-type lambda is not very lytic, compared to coli phages T4 or T7.

E. coli can easily overgrow phage lambda in liquid culture, but

recombinant lambda phage show very good growth on large (150-mm

diameter) plates, rather than in liquid culture because Recombinant

lambda phage are usually less lytic than wild-type.

The host for lambda is E. coli C600 and its subsequent variants. To

avoid complications, the E. coli C600 strain is usually restriction-

negative: r-m- or r-m+. E. coli host for lambda is grown on LB (and

many other) medium, and E. coli growth is faster.

The lambda receptor on the bacterial cell outer surface is part of the

maltose-usage pathway. To induce the receptor to high levels (102-103

55
receptors per cell), maltose is added to a final concentration of 0.2%

in the medium.

When growing lambda on plates to prepare DNA (as opposed to

picking plaques or titering), agarose is substituted for agar. Agarose is

much more expensive than agar, but does not contain impurities that

inhibit enzymes. Lambda DNA prepared from agar plates may not be

digestible by restriction enzymes because of the presence of inhibitors

that have leeched from the agar.

It was useful to start the culturing with a fresh E. coli colony on LB

plates. The colony was picked into 5 ml of LB medium plus 0.2%

maltose and placed on a wheel at 37°C for approximately 4 hr. This

time would be much longer if the colony will from an old plate or if it

stored at 4°C or frozen. Under sterile conditions, the culture was

transferred to a sterile 15-ml conical tube. The E. coli were pelleted by

centrifugation in an SS34 rotor with adaptors in an RC-5B centrifuge

for 8 min at 4 K rpm . The medium was poured off gently. The

bacterial pellet was resuspended in 4-6 ml of 0.01 M MgSO4 by

pipette up and down using sterile conditions, but not by vortexing.

The concentration of bacteria was adjusted to yield an "optical

56
density" of 2.0 at wavelength 600 nm. E. coli prepared in this way

was stored at room temperature and used for our work. Cells prepared

in this manner were employed at values of 10 µl per small or 20 µl per

large plate. (Sambrook, et al.,1991) Sambrook, Fritsch, and Maniatis,

in "Molecular Cloning: a laboratory manual", pp. 2.60-2.79, second

edi tion, 1991, as the "CSH" procedure or the "CSH" book. SM

buffer was used Lambda phaga was growm on "SM buffer" prepared

with Tris buffer with magnesium salt and NaCl.

The isolated component was confirmed as Phage lambda DNA in

Agarose gel electrophoresis by using the Lambda DNA marker(Fig-

1)

The isolated components from the phage lambda were kept in 8

agarose wells and run in agarose gel electrophoresis and then

confirmed as DNA fragments through the agarose electrophoresis gel

bands(Fig-2).

The amplified PCR products were separated into four sets and each

set containing two amplified PCR products for the purification.

57
Now in each set of PCR amplified DNA the two samples were

purified in two ways

1) Gel purification and

2) Direct purification.

In a set, one sample was subjected to 2% agarose gel

electrophoresis, and then cut the gel pieces contain DNA bands were

purified through four different selected methods. And the second

sample was directly purified without electrophoressed. The direct

purification method follows the same protocol as gel purification

protocol except gel electrophoresis.

We selected the better available methods for purification of

amplified PCR products.

1) Phenol, high salt precipitation method

2) Spin column method

3) Silica gel method and

4) Enzyme method (Novel new method – recently

introduced method)

In this study we had carried out direct sequencing of PCR

products as this approach involves fewer steps, thereby, the faster and

cheaper sequence data is obtained. It was found that the PCR product

58
used in the sequencing was very well purified by using 仇 micro filter

spin unit system (Millipore) resulting in good sequence data. However

the sequence data of PCR product, purified using Quick Spin Column

was not satisfactory. This was probably due to the need of care.

Manipulation steps which are crucial when the columns were being

used. If the, PCR product is pure and free from miss primer or

nonspecific amplification products, clear sequencing ladders will

usually result (Meltzer, 1993).

We sent our samples to MWGAG Biotech, Bangalore to assign

the DNA sequence to reveal the sequence and it gave the sequencing

results as follows.

Unpurified sample

To have an understanding about the comparison of purified and

unpurified DNA samples, we had sent an unpurified sample and got

the sequence analysis. The resultant graph(Fig-3) was saying that the

unpurified sample had so many number of the nucleotide sequences

but actually Phage lambda DNA has no that much lengthy nucleotide

sequence. It meant that the unpurified PCR amplified product has

somany impurities including primer sequences that were used in PCR

amplification.

59
Purification results of the PCR amplified amplified

products (in protocols wise)

1) Purified sample by Phenol mix protocol

The Agarose gel eluted DNA was purified by using

phenol mix method and it was sent for sequencing for checking the

purity of eluted DNA. By using the phenol mix method the purity

was determined as 25% for direct purified sample and 20% for gel

purified sequence

(Fig-4).

This is very common conventional method that used to purify

PCR amplified products. This involved that mixing PCR products

with phenol reagent mix. After brief vortex, three layers were formed

where pure DNA alone stands on the top of the solution and

contamination like Polymerase enzyme, proteins, buffers, chemicals,

Mg ions, primers were trapped in phenol.

Supernatant having DNA was mixed with chloroform to

remove residual effects of phenol and precipitated with iso proponal.

DNA pellet was extensively washed with 70% ethanol to remove any

60
co precipitated chemicals and finally DNA pellet was dried and

suspended with Distilled water.

Disadvantages; (Justified as per our three conditions)

Duration to perform the experiment; It took more than 2 and half

hours and laborious protocol.

Purity percentage was 25% (Most of time, we lost our DNA,

because of very minute quantity, not formed pellet, which makes

scientists to confuse with this purifications.

Cost of chemicals: Even though it is cheaper, it is laborious.

Others:

• This is laborious and need much care to prepare buffers and

reagents.

• Phenol is listed as dangerous and potential chemicals for health.

Even a small spill makes worst to skin and with characteristic odor

cause epithelial cells to damage.

61
2) Purified sample by Silica gel method

The eluted DNA was purified by using Silica gel method

and it was sent for sequencing for checking the purity of eluted DNA.

By using the Silica gel method the purity was determined as 50% in

direct purification method and only 40% in gel purification

method(Fig-5).

This method was followed by very few industries in India

in 1980’s because of the moderate expense, but the purification and

the recovery of PCR product from the Agarose gel takes longer

time ,nearly 16 hours, because after the silica gel is prepared, it has

to kept incubation for over night, then only it should used for PCR

mixing.

3) Purified sample by Spin Column Methods

The eluted DNA was purified by using spin columns and it

was sent for sequencing for checking the purity of eluted DNA. By

using the spin column method the purity was determined as more

than 70 % for both direct purification and gel purification

samples(Fig-6).

Interpretation (based on three factors)

62
Duration to perform the experiment for direction purification- 45min

and

For gel based purification- 1 hour 45min.

Purity percentage is more than 70% (with reference to sequencing

reports)

Chemicals are Costly, we have bought from commercial kit

manufacturers .

4) Purified sample by Enzymatic method

The eluted DNA was purified by using Enzymatic method and

it was sent for sequencing for checking the purity of eluted DNA. By

using the Enzymatic method , the purity was determined as more than

85% for the direct purified sample and more than 80% for the gel

purified sample(Fig-7).

The time consumed for this method is very less , it is only 20

minutes for the direct purified sample and 1 hour 45 min for the gel

63
purified sample. In this method we used Calf intestine alkaline

phosphatase enzyme which was responsible for recovery of highly

purified PCR amplified product from the agarose gel. We felt it was

the best method for PCR amplified purification.

If we observe all the methods of purification of PCR amplified

product at a glance (Tab-1)the mathematical figures are revealing that

the enzymatic method is the best method in all angles because it is

consuming only 90 min, cost is moderate and the labour is less.

Out of four methods, we found enzymatic method (Mandelkern

M, et al., 1981) and spin column method (Butler, et al .,2001)are best

methods, however, we suggest enzymatic method is most suitable for

researchers, because its takes only 30 minutes and single tube work

and the cost of the purification is also moderate.The other two

methods are laborious and yield of purified DNA is lesser.

64
 Duratio Purit
Methods n y Cost
Phenol Mix method - Direct
purification 2 hours 25% Cheaper
Phenol Mix method - Gel
separated DNA 4 hours 20% Cheaper
Silica gel method - Direct
purification 8 hours 50% moderate
Silica gel method - Gel separated 16hour
DNA 30min 40% moderate
Spin column method - Direct
purification 45min >70% Costly
Spin column method - Gel 1hour
separated DNA 45min >70% Costly
Enzyme method - Direct
purification 20min >85% moderate
Enzyme method - Gel separated 1hour
DNA 20min >80% moderate

Table-1 Different purification protocols and their conclusion

65
Fig-2 Agarose gel electro phoresis

Fig- 3 Unpurified sample sequence

66
Fig-4 Phenol mix purified sequence

Fig-5 Silica gel purified sequence

67
Fig-6 Spin column purified sequence

Fig-7 Enzymatic purification method

68
 SUMMARY AND CONCLUSION

Molecular biology deals with the DNA, RNA and genes.

Among them genes are responsible for expression of characters.

Every gene has its own expression. Some times when we need a new

gene expression from the existing genes, they have to be manipulated.

The manipulation involves isolation of DNA, isolation of gene of

interest, amplification of gene, hybridization of genes and insertion of

newly constructed gene into an organism for expression.

For cloning of an amplified gene, the DNA coding for the gene

should be highly purified and it should be in excess quantity to over

express the character. In most cases, the researchers are failing to

clone a desired gene which was eluted from the agarose gels because

of impurities present in the isolated genes. Here we are concentrating

on the best method which gives maximum elution of (75%-80%)

DNA from the agarose gels.

Though thousands of the methods are available for the recovery

of purified PCR products, some are time consuming, some are costly

and some are giving poor yield of purified products and some are

laborious. To over come all the above problems and to provide an

easy, rapid, low cost and high amount of purified PCR products there

69
should be a novel method. The present study aims this as a challenge

and concentrated on the best method which can solve all the above

mentioned problems. To achieve this, 4 best protocols among the

available methods and proceeded according to their instructions.

The selected protocols were

1) Phenol mix method,

2) Silica gel method,

3) Spin column method and

4) Enzymatic method.

The Lambda phage DNA was confirmed in 1.2% Agarose gel

electrophoresis. Then the template DNA was subjected for PCR

amplification. By using this template DNA PCR amplification was

proceed. In this contest the gene was amplified by PCR and the 500bp

product was evolved. The amplified product was electrophoresed

along with 1Kb molecular weight marker in 1.8% agarose. After

confirmation of optimized PCR amplification protocol the DNA was

eluted from gels and it was send for DNA sequencing. By using this

sequencing process the amplified gene was confirmed and it reports

the perfect elution of DNA by enzymatic method. We sent our

70
samples to MWGAG Biotech, Bangalore and it gave the sequencing

results.

The sequencing results in the form of the graphical

representations revealed that the enzymatic purification method was

the best method for PCR amplified product purification. The recovery

of the purified PCR product was 85% in direct purification and it was

more than 70% in gel purified product. The enzyme alkaline

phosphatase used in this method gave the high percentage of

purification and the time taken for that was only 20 min for direct

purification and 1hour 20 min for gel purification. The expenditure

was also moderate. Silica gel method and Phenol mix method were

time taken, and gave less than 50% of the purified PCR product.

Though the Spin column method gave nearly 70% of the Purified

product and the time was consumed 2hours and 30 min, the cost is

high, because the chemical expenditure is high. We use phenol in

phenol mix method but Phenol is not safe. Silica gel method was very

laborious and time taken. Even the preparation of silica gel had taken

nearly 12 hours and the cost of silica gel is high. In view of all these

considerations we decided the Enzymatic method is the best suitable

method to purify the PCR amplified product.

71
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