GENERAL AND SPECIALIZED METHODS AND TECHNIQUES IN MICROBIAL

GENETICS

In 1953 American geneticist James Watson and English physicist Francis Crick

discovered the double-helical structure of DNA. Discovery of the double-helical structure was

the key that enabled Watson and Crick to learn how DNA is replicated.

In the late 1950’s, Matthew Meselson and Franklin Stahl first described the DNA

molecule and how DNA replicates in a process called semi conservative replication because each

daughter molecule comprises one parental strand and one newly synthesized strand. The parental

double helix of DNA unwinds and each strand act as a template for the production of a new

complimentary strand.

DNA REPLICATION IN PROKARYOTES

Bacteria multiply by a process of binary fission; before this occurs, each cell must

duplicate its genetic information so that each daughter cell has a copy.

DNA replication involves the action of a number of specialized enzymes:

- Helicases

- DNA topoisomerases

- DNA polymerase I

- DNA Polymerase III

- DNA primase

- DNA ligase

Replication begins at a specific sequence called an origin of replication. The two strands

of DNA are caused to separate (unwind) by helicases while single-stranded DNA binding

proteins prevents them rejoining. Opening out part of the double helix causes increased tension
(supercoiling) elsewhere in the molecule, which is relieved by the enzymes DNA topoisomerase

(sometimes known as DNA gyrase). As the zipper moves along, and more single stranded DNA

is exposed, DNA polymerase III adds new nucleotides to form a complementary second strand.

According to the rules of base pairing DNA polymerases are not capable of initiating the

synthesis of an entirely new strand, but can only extend an existing one. This is because they

require a free 3’ – OH group onto which to attach new nucleotides. Thus, DNA polymerase III

can only work in the 5’ – 3’ direction. A form of RNA polymerase called DNA primase

synthesizes a short single strand of RNA, which can be used as a primer by the DNA polymerase

III.

When replication occurs, complementary nucleotides are added to one of the strands (the

leading strand) in a continuous fashion. The other strand (the lagging strand), however runs in

the opposite polarity. A complementary sequence is synthesized when DNA polymerase III

allows a little unwinding to take place and then, starting at the fork, works back over from a new

primer, in the 5’ -3’ direction. Thus, the second strand is synthesized discontinuously, in short

bursts, about 1000 – 2000 nucleotides at a time. These short stretches of DNA are called Okazaki

fragments, after their discoverers.

On the lagging strand, a new RNA primer is needed at the start of every Okazaki

fragments. These short sequences of RNA are later removed by DNA polymerase I, which then

replaces them with DNA nucleotides. Finally, the fragments are joined together by the action of

DNA ligase.
 lagging strand
5’ 3’
Single stranded
binding protein DNA DNA
Primase Polymerase
e 5’ - 3’
helicase
RNA primer Okazaki
5’ Fragment
3’
DNA polymerase III
helicase 5’
3’

3’ 5’
Leading strand

Fig 1: Okazaki fragments

POLYMERASE CHAIN REACTION (PCR)

The PCR invented by Kary Mullis in the early 1980’s exploded onto the biotechnology

landscape. It has had such a profound impact on biology, biochemistry and medicines. It is

probably the most significant development in molecular biology since the advent of gene

cloning. PCR is important because it enables the rapid synthesis of many, many copies of a

specific DNA fragment from a complex mixture of DNA. Researchers can thus obtain large

quantities of specific pieces of DNA for experimental and diagnostic purposes.

To make large quantities of a particular sequence, a process known as DNA or gene

amplification. The first step is to synthesize DNA fragments with sequence identical to those

flanking the targeted sequence. This is accomplished with a DNA synthesizer. The synthetic

oligonucleotides are usually about 20 nucleotides long and serve as DNA primers for DNA
synthesis. The primers are one component of the reaction mixture, which also contains the target

DNA, a thermostable DNA polymerase and each of the four deoxyribonucleoside triphosphates

(dNTPs). PCR requires a series of repeated reactions, called cycles. Each cycle has three steps

that are precisely executed in a machine called a themocycler.

The three steps in the PCR are:

Denaturation

By heating to 95ºC the DNA is separated into single strand providing a template for the

DNA polymerase.

Primer annealing

The temperature is lowered (typically 50ºC - 60ºC) depending on the primer sequence so

that the primers can hydrogen bond or anneal to the DNA on both sides of the target sequence.

Because the primers are very small and are present in excess, the targeted DNA strands anneal to

the primers rather than to each other.

Extension

At around 72ºC, DNA polymerase extends the primers and synthesizes copies of the

target DNA sequence using dNTPs. Taq polymerase from the thermophilic bacterium Thermus

aquaticus is used in the PCR technique. The optimum temperature for Taq polymerase is 72ºC,

and it can tolerate being raised to values considerably higher than this for short periods. At the

end of one cycle, the targeted sequences on both strands have been copied. When the three-step

cycle is repeated, the two strands from the first cycle are copied to produce four fragments.

These are amplified in the third cycle to yield eight double-stranded products. Thus, each cycle

increases the number of target DNA molecules exponentially. Depending on the initial

concentration of the template DNA and other parameters, it is theoretically possible to produce
about 1 million copies of targeted DNA sequence after 20 cycles and as many as 1 billion after

30 cycles. Typical PCR protocols run for 30 – 35 cycles. All this can be achieved in just a couple

of hours in a thermocycler

PCR is an essential tool in many areas of molecular biology, medicine, and biotechnology. When

PCR is used to obtain DNA for cloning, a number of steps traditionally employed are no longer

required. PCR is also used to generate DNA for nucleotide sequencing. Because the primers used

in PCR target specific DNA, PCR can isolate specific fragments of DNA (e.g., genes) from

solutions that contain many different genomes such as soil, water, and blood. For instance, PCR

is used in a number of diagnostic tests, including those for AIDS, Lyme disease, chlamydia,

tuberculosis, hepatitis, human papillomavirus, and other infectious agents and diseases. The tests

are rapid, sensitive, and specific. PCR is particularly valuable in detecting genetic diseases such

as sickle cell anemia, phenylketonuria, and muscular dystrophy. The technique is also employed

in forensic science, where it is used in criminal cases as part of DNA fingerprinting technology.

It is possible to exclude or incriminate suspects using extremely small samples of biological

material discovered at the crime scene.

GEL ELECTROPHORESIS

Gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA separation are

often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When

the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a

solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that

are held together by hydrogen bonds and form tiny pores.

are placed. Before the DNA samples are added, the gel must be placed in a gel box. One end of

the box is hooked to a positive electrode, while the other end is hooked to a negative electrode.

The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution

that can conduct current, the buffer fills the gel box to a level where it just barely covers the gel.

The end of the gel with the wells is positioned towards the negative electrode. The end without

wells (towards which the DNA fragments will migrate) is positioned towards the positive

electrode.

Once the gel is in the box, each of the DNA samples to be examined (for instance, each PCR

reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One

well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known

lengths.

Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA

molecules have a negative charge because of the phosphate groups in their sugar-phosphate

backbone, so they start moving through the matrix of the gel towards the positive pole.

As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than

longer ones. After the gel has run for a while, the shortest pieces of DNA will be close to the

positive end of the gel, while the longest pieces of DNA will remain near the wells. Once the

fragments have been separated, the gel can be examined and see what sizes of bands are found

on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA

fragments will glow.

DNA fragments of the same size that have all traveled as a group to the same position. A single

DNA fragment (or even a small group of DNA fragments) would not be visible by itself on a gel.

By comparing the bands in a sample to the DNA ladder, we can determine their approximate

sizes.

All DNA molecules have the same amount of charge per mass. Because of this, gel

electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we

can see how many different DNA fragments are present in a sample and how large they are

relative to one another.

1. Conventional PCR

The polymerase chain reaction (PCR) is a test tube system for DNA replication which allows a

“target” DNA sequence to be selectively amplified several million folds in just a few hours. PCR

enables the synthesis of specific DNA fragments using a DNA-polymerase enzyme, which takes

part in the replication of the cellular genetic material. This enzyme synthesizes a complementary

sequence of DNA, as a small fragment (primer) is connected to one of the DNA strands in the

specific site chosen to start the synthesis.

Primers limit the sequence to be replicated, and the result is the amplification of a particular

DNA sequence with billions of copies. Conventional PCR is applied in selective DNA isolation,

amplification and quantification of DNA, medical and diagnostic approaches, infectious disease

diagnosis, forensic studies and research areas.

2. Multiplex PCR

Multiplex PCR is a common molecular biology technique used for the amplification of multiple

targets in a single PCR test run. Multiple primers and a temperature-mediated DNA polymerase

are used for the amplification of DNA in a thermal cycler.

All the primers pairs designed for Multiplex PCR have to be optimized so that the same

annealing temperature is optimal for all the pairs during PCR. When multiple sequences are

targeted at once, additional information can be generated from a single test run which otherwise

would require a larger amount of the reagents and extensive time and effort to perform.

This technology has been applied in many areas such as genotyping, mutation and polymorphism

analysis, detection of pathogens or genetically modified organisms, etc. In diagnostic

types of diseases.

3. Nested PCR:

It is a useful modification of PCR technology where the specificity of the reaction is enhanced by

preventing the non-specific binding with the help of the two sets of primer. The first set of

primer binds outside of our target DNA aIn the second round of amplification, second set of

primer amplifies only the target DNA.

Nested PCR is a helpful method for the phylogenetic studies and detection of different

pathogens. The technique has higher sensitivity; hence even if the sample contains lower DNA, it

can be amplified which is not feasible in the conventional PCR technique.

4. Amplified Fragment Length Polymorphism (AFLP) PCR

It is a PCR-based technique that uses selective amplification of a section of digested DNA

fragments to generate unique fingerprints for genomes of interest. This technique can quickly

generate large numbers of marker fragments for any organism, without prior knowledge of the

genomic sequence.

AFLP PCR uses restriction enzymes to digest genomic DNA and allows attachment of adaptors

to the sticky ends of the fragments. A part of the restriction fragments is then selected to be

amplified by using primers that are complementary to the adaptor sequence.

AFLP PCR is employed for a variety of applications, as to assess genetic diversity within species

or among closely related species, to infer population-level phylogenies and biogeographic

patterns, to generate genetic maps and to determine relatedness among cultivars.

5. Reverse Transcriptase PCR (RT-PCR)

molecules are first converted into complementary DNA (cDNA) molecules that can then be

amplified by PCR.

In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using

reverse transcriptase. The cDNA then acts as a template for exponential amplification using

PCR.

RT-PCR can be conducted either in a single tube or as two steps in different tubes. The one-step

method is more effective with fewer chances of contamination and incorporation of variations.

RT-PCR is used in research methods, gene insertion, genetic disease diagnosis and cancer

detection.

6. Real-Time PCR (Quantitative PCR (qPCR))

Quantitative PCR (qPCR), also called real-time PCR or quantitative real-time PCR, is a PCR-

based technique that couples amplification of a target DNA sequence with quantification of the

concentration of that DNA species in the reaction.

Conventional PCR is a time-consuming process where the PCR products are analysed through

gel electrophoresis. qPCR facilitates the analysis by providing real time detection of products

during the exponential phase. The principle of real-time PCR depends on the use of fluorescent

dye. The concentration of the nucleic acid present into the sample is quantified using the

fluorescent dye or using the fluorescent labelled oligonucleotides.

q-PCR is applied in genotyping and quantification of pathogens, microRNA analysis, cancer

detection, microbial load testing and GMOs detection.

8. Randomly Amplified Polymorphism DNA (RAPD)

9. Restriction Fragment Length Polymorphism (RFLP) PCR

10. Reverse Transcriptase Real-Time PCR (RT-qPCR)