Techniques employed in recombination DNA technology: Isolation and purification of DNA, Gel electrophoresis (Gel, Agarose), DNA sequencing, Southern and Northern blotting, PCR and its application

Isolation and purification of DNA:-

1. The extraction buffer:- This includes -

i. Detergent:- Such as cetyl trimethyl ammonium bromide(CTAB) or SDS which disrupts the membranes.

ii. Reducing agent:- Such as Beta mercaptoethanol which helps in denaturing proteins by breaking the disulfide bonds between the cysteine residues and for removing the tanins and polyphenols present in the crude extract.

iii. Chelating agent:- Such as EDTA which chelates the magnesium ions required for DNase activity.

iv. Buffer:- which is almost always Tris at pH 8.

v. Salt:- Such as sodium chloride which aids in precipitation by neutralizing the negative charges on the DNA so that the molecules can come together. 

2. Phenol chloroform extraction:- 

> Nucleic acid solutions commonly contain undesirable contaminants that are chiefly made of proteins. A classic method of purifying is phenol –chloroform extraction by which the the nucleic acid solution is extracted by successively washing with - 

i. a volume of phenol(pH 8.0)

ii. a volume of phenol: chloroform: isoamyl alcohol (25: 24:1)

iii. a volume of chloroform: isoamyl alcohol ( 24:1)

> Centrifugation is performed intermittently and the upper aqueous phase is transferred to a new tube while avoiding the interphase. 

> The contaminants are denatured and accumulate in the organic phase or in the marginal layer between the two phases and the nucleic acids are preserved in the aqueous phase. 

> Another way of removing proteins is by using the enzyme proteinase K which however again is denatured by phenol via phenol chloroform extraction. 

3. Precipitation of nucleic acids:- 

> Alcohol precipitation is the most commonly used method for nucleic acid precipitation. 

> This requires diluting the nucleic acid with a monovalent salt, adding alcohol to it and mixing gently. 

> The nucleic acid precipitated spontaneously and can be pelleted by centrifugation. 

> The salts and alcohol remnants are removed by washing with 70% alcohol. 

> The most commonly used salts include:-

i. Sodium acetate pH 5.2 (0.3M)

ii. Sodium chloride (0.2M)

iii. Ammonium acetate (2- 2.5M)

iv. Lithium chloride (0.8M) 

v. Potassium chloride. 

> Ethanol (twice the volume) or isopropanol ( two thirds volume) are the standard alcohols used for nucleic acid precipitation. 

4. Resuspending DNA:- The nucleic acid pellet can be resuspended in either sterile distilled water or TE(10 mM Tris:1mM EDTA) 

5. Purification of DNA:- The DNA is purified by incubating the nucleic acid solution with RNase A (10mg/ml) at 37° C and reprecipitation following phenol: chloroform extraction to remove the RNase. 

Gel Electrophoresis:-

> This technique is a used to separate macromolecules such as DNA, RNA, and proteins.

> Macromolecules are separated based on their size and electric charge. 

Agarose Gel:- 

- The porous gel is used which acts as a molecular sieve.

- The gel is made of a material called agarose, which is a gelatinous substance extracted from seaweed Gelidium. 

- The gel is submerged in a salt buffer solution in an electrophoresis chamber. 

- Tris-borate-EDTA (TBE) is commonly used as the buffer. Its main function is to control the pH of the system. 

- Samples are then loaded into tiny wells in the gel with the help of a pipette.

Charged Electrodes:-

- The chamber has two electrodes – one positive and another negative - at its two ends.

- Two oppositely charged electrodes pull molecules towards them on the basis of their charge.

- An electrical current of 50–150 V is applied.

- On application of electric charge, smaller and strongly charged molecules move faster across the gel while the larger and weakly charged molecules move slower and left behind.

Visualization of bands:-

- Once the separation is complete, the gel is stained with a dye to reveal the separation bands. 

- Ethidium bromide is a fluorescent dye commonly used in gel electrophoresis. 

- The gel is soaked in a diluted ethidium bromide solution and then placed on a UV transilluminator to visualize the separation bands.

- The bands are immediately examined or photographed for future reference, as they will diffuse into the gel over time.

Applications:-

- In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes.

- To analyze results of polymerase chain reaction.

- To analyze genes associated with a particular illness.

- In DNA profiling for taxonomy studies to distinguish different species.

- In paternity testing using DNA fingerprinting.

- In the study of structure and function of proteins.

- In the analysis of antibiotic resistance.

- In blotting techniques for analysis of macromolecules.

- In the study of evolutionary relationships by analyzing genetic similarity among populations or species.

DNA sequencing:-

> It is the process of determining the sequence of nucleotides in a piece of DNA.

> Methods:-

a. Maxam - Gilbert method

b. Sanger's method

a. Maxam - Gilbert method:-

- Also called as chemical cleavage method.

- Discovered by American molecular biologists Allan M. Maxam and Walter Gilbert in 1973.

Procedure:- The steps of Maxam Gilbert Sequencing are: 

i. Radioactive labeling of the 5′ end by a kinase reaction using gamma-32P ATP and purification.

  

ii. Four chemical Treatments for A+ G,  G, C + T, C bases (Depurination of purines (A+G) by  formic acid, Methylation of guanine (G) by  dimethyl sulfate, Hydrolyization of pyrimidines  (C+T) by hydrazine, and Inhibition of the hydrazine  reaction for thymine by the addition of sodium chloride, hydrolyzing  only cytosine (C).

iii. Cleavage of the modified DNA by hot piperidine;  (CH2)5NH  at the position of the modified base producing a series of labeled fragments from the radiolabeled end to the first ‘cut’ site.  

iv. Size fractionation of the fragments on a PAGE (polyacrylamide gel electrophoresis).  

v. Visualization by autoradiography, inferring the sequence.

b. Sanger's method:-

- Also called as dideoxy method.

- Discovered by English biochemist Frederick Sanger in 1977. He got Nobel Prize in Chemistry in 1980.

Procedure:- The Sanger sequencing method consists of 6 steps:

i. The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA).

ii. A primer that corresponds to one end of the sequence is attached.

iii. Four polymerase solutions with four types of dNTPs but only one type of ddNTP are added.

iv. The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated.

v. The resulting DNA fragments are denatured into ssDNA.

vi. The denatured fragments are separated by gel electrophoresis and the sequence is determined.

Blotting:-

> It is the separation and transfer of macromolecules onto a blotting membrane to detect and identify such macromolecules, as DNA, RNA or Protein.

> 4 types:-

i. Southern blotting:- Detect DNA.

ii. Northern blotting:- Detect RNA.

iii. Western blotting:- Detect Protein.

iv. Eastern blotting:- Detect Protein.

General Procedure:-

- The target molecule in a sample is isolated.

- Electrophoresis separates the molecules.

- The separated contents are transferred onto a membrane. This process is celled blotting.

- The membrane is then exposed to radiolabeled probes and incubated. Probe binds to the target molecule.

- The probe and target molecule create bands that can be visualized with X-ray film.

1. Southern blotting:-

- Discovered by Edward M. Southern at Edinburgh University in 1970.

- It is designed to locate a particular sequence of DNA within a complex mixture. For example, it could be used to locate a particular gene within an entire genome.

Procedure:-

a. Digestion:- Digest the DNA with an appropriate restriction enzyme.

b. Electrophoresis:- Run the digest on an agarose gel.

c. Denaturation:- Denature the DNA on the gel. For this soak it in about NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.

d. Depurination:- Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl takes the purines out, cutting the DNA into smaller fragments. Neutralize the acid after this step.

e. Blotting:- 

- It is the transfer the denatured DNA to the membrane.

- A nitrocellulose membrane or nylon membrane is used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm.

- Many scientists feel nylon is better since it binds more and is less fragile.

- Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.

- You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC (Saline Sodium Citrate) through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)

- After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)

f. Hybridization:- 

- It is the process of annealing the probe to the DNA on the membrane due to complementarity.

- Probing is often done with:

i. 32P labeled ATP

ii. Biotin / Streptavidin

iii. Bioluminescent probe

g. Visualization:- 

- Visualize your radioactively labeled target sequence. 

- If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. 

- Biotin / Streptavidin detection is done by colorimetric methods.

- Bioluminescent visualization uses luminesence.

2. Northern Blotting:-

- The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. 

- Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.

- It is designed to locate a particular sequence of RNA within a complex mixture.

Procedure:-

a. Extraction of RNA:- There are many RNA extraction kits commercially available, but they all involve cell lysis, inhibition of RNAases, removal of proteins and other contaminants, and recovery of RNA.

b. Isolation of mRNA:-

- Oligo dT cellulose chromatography can be used to isolate only mRNA with a polyA tail. The poly A tail is the final step of mRNA production in the nucleus. The tail enables nuclear export, translation, and stability of mRNA. 

- In Oligo dT cellulose chromatography, oligos complementary to the poly A tail are covalently attached to a resin column. 

- When the sample is applied to the column the mRNA with the poly A tail will hybridize to the oligo probe and be retained on the column. 

- Then, the elution buffer is applied to disrupt hybridization and recover the mRNA.

c. Gel electrophoresis to separate mRNA by size:-

- Agarose gels containing formaldehyde were traditionally used to denature RNA. 

- The formaldehyde reacts with the imine and amine groups on the nucleic acids, which disrupts the hydrogen bonding between bases and disrupts the secondary structure of the RNA. 

- It is important to disrupt the secondary structure because the RNA must be extended to allow proper binding of probe for identification.

d. Transfer of RNA to blotting membrane:-

- The transfer is necessary because the probes can’t enter into the gel matrix. Therefore, the RNA must be transferred to a membrane where they can be accessed by the probes.

- Transfer is accomplished via a capillary (overnight) or vacuum (15-60 minutes) blotting system.

- The blotting membrane is positively charged to attract the negatively charged RNA. Nylon is a commonly used membrane.

v. Immobilization of RNA to the blotting membrane:- Covalently attached to the membrane by the application of UV light or heat.

vi. Application of Probe:- 

- Probes have a minimum of 25 bases that are complimentary to the mRNA sequence of interest.

- Excess probe is washed off.

vii. Probe visualization:-

- Radioactive isotopes were traditionally used, but have been replaced in favor of safer detection methods.

- Chemiluminescence is commonly used in the modern northern blot protocol.

PCR and its application:-

•  PCR:- The process of multiplication of DNA segments using DNA polymerase and DNA primers is called PCR.

•  Discovery:- The PCR technique was discovered by Kary Mullis in 1985.

•  Thermocyclers are used to achieve different temperatures.

•  PCR has three main steps -

i. Denaturation:- When dsDNA is heated, both its chains separate at a temperature of 90°C.

ii. Primer Annealing:- Primers are attached at the 5 'end of single chains at a temperature of 55°C.

iii. Polymerization:- DNA polymerase enzyme polymerize the primers at 70°C temperature.

•  Heat stable DNA polymerase:-

Ø Normal DNA polymerase is heat sensitive. It is destroyed due to its deformation at 90°C temperature. Therefore, we cannot use normal DNA polymerase in PCR.

Ø Instead, we use heat stable DNA polymerase in PCR which can tolerate high temperature and does not deform.