Real time PCR

Real time PCR

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The real time polymerase chain reaction is also called as q-PCR (quantitative PCR).

It is a laboratory technique based on PCR, which is used to amplify and simultaneously quantify the targeted DNA molecule.

Its key feature is that the amplified DNA is detected as the reaction proceeds in real time.

The common methods:

1.       Use of non-specific dyes: the nonspecific dyes are not complementary to any specific sequences of the DNA, rather it intercalates with the double stranded DNA.

2.       Sequence specific fluorescent probes: consists of oligonucleotides that are labeled with fluorescent reporters, which permits detection only after hybridization of probe with targeted DNA.

3.       The probe is tagged with fluorescent materials (reporter) along with is attached the quencher molecule in very close proximity with the reporter. The quencher quenches (absorbs) the fluorescence of the reporter.

4.       The probe is first hybridized with the DNA, then the polymerase is allowed to perform its task, as the DNA polymerase adds nucleotides to the template strand, the flurophore is released away from the quencher molecule and the fluorescence is produced, depicting the formation of double stranded DNA. The intensity of fluorescence is directly proportional to the quantity of the ds DNA formed.

PCR: polymerase chain reaction.

·         PCR is a very simple process.

·         All that happens in PCR is a short region of DNA molecule, let’s take for example a single gene, and is copied many times by DNA polymerase enzyme.

·         The PCR has a variety of applications in genetics, research and in broader areas of biology.

An outline of polymerase chain reaction:

·         It results in selective amplification of chosen reaction of the DNA molecule.

·         For amplification, a region of DNA is chosen (whose sequences in border regions are known)

·         The border sequences of the DNA fragment to be amplified must be known, because in order to carry PCR, two short oligonucleotides must hybridize to the DNA molecule, one to each strand of double helix.

·         These oligonucleotides are used as DNA primers for DNA synthesis reaction.

·         Amplification is usually carried out by DNA polymerase I enzymes derived from thermus aquaticus (this bacteria inhabits the hot streams)

·         The polymerase is named taq polymerase after the name of bacteria from which it is derived.

·         Taq polymerase is thermostable and can withstand temperature up to 96 ⁰ C.

·         The PCR is a very sensitive technique; it can even start from a single target molecule.

·         The size of DNA that can be amplified by this technique is 10 to 40 Kb.

Components required to carry out polymerase chain reaction (PCR):

·         DNA template (with known end sequence)

·          Primers ( the primers complementary to the known sequence of target DNA molecule,

·          taq DNA polymerase (isolated from bacteria thermus aquaticus) living in hot springs. It can withstand temperature up to 96 ⁰ C),

·          fixed buffer( to maintain favourable environment during the PCR),

·          Divalent cations (Mg²⁺ is used in general. Mn²⁺ can be also used, but at higher concentration it causes mutation),

·          monovalent cations (K⁺ is used in general)

·         Large number of DNA nucleotides

Procedures of PCR:

·         The PCR consists of 20 to 40 thermal cycles.

·         Each cycles has discrete steps

1.       Hold: the cycle starts with a temperature of 96 ⁰ C. the hold lasts for a brief period.

2.       Initializing step: the temperature is raised further to 94 to 96 ⁰ C. (if the DNA polymerase to be used is highly thermostable then the temperature can be raised up to 98 ⁰ C.)

This step lasts up to 1 to 6 minutes.

3.       Denaturation step: at the 94⁰ C to 96 ⁰ C the tubes containing the target DNA is placed in the machine.

At this temperature the DNA melts—the strands get separated by breading of hydrogen bonds. At the end of the denaturation step the result is the 2 separated ssDNA molecules.

4.       Annealing step: in this step the temperature is decreased down to 50 to 60 ⁰ C and primers are added and are carried out for 20 to 40 seconds.

Also the DNA polymerase is added too. The primers pair with the complementary sequences on target DNA molecules.

Note: annealing temperature should always be 2 to 3 ⁰ C less than melting temperature of primers, to prevent the primers from melting down.

At this temperature H-bonds are formed and DNA polymerase binds to end of primers.

5.       Elongation: at this step the temperature is raised to 70 to 75 ⁰ C.

At this temperature taq DNA polymerase acts at its best (best temperature is 72 ⁰ C)

6.       Final elongation: at this step the temperature is maintained from 70 to 74 ⁰ C for 5 to 15 minutes after last cycle of PCR, to ensure that all last DNA are fully extended.

 

Plasmid vectors: a brief study

Plasmid vectors: a brief study _______________________________
Presented by:
MANOJ KUMAR
SUKUMAR DANDPAT
VI YEAR__________________________________________________________________________________________________________________________

Genetic engineering stands for the transfer of DNA between hosts (or the species) by in vitro enzymatic manipulation. This means that the DNA to be transferred will be duplicated in the new host. Since most DNA fragments are incapable of self replication in E.coli or any other host cell, an additional segment of the DNA, capable of autonomous replication must be linked to the fragment to be cloned. This autonomously replicating segment is the molecular cloning vectors.
Thus vectors are self replication DNA molecules into which foreign DNA molecules are inserted (Gardner et al. 2001). Vectors have been developed and used in delivery of foreign genes into cells and chromosomes of prokaryotes and eukaryotes. Such a vector plays a central role in recombinant DNA technology.
Most cloning vectors are originally derived from naturally occurring extrachromosomal elements such as bacteriophage and pladmids.
Stanley cohen and his coworkers (1975) first reported the use of bacterial plasmids as molecular cloning vectors. Since that initial report, thousands of cloning vectors have been constructed, and their versatility in terms of cloning sites, host-range and function appears to be limited only by the deftness and imagination of inventor.
Currently specialized cloning vectors are available that enable the investigator to:
1. Identify and isolate regulatory DNA sequences such promoters and terminators;
2. Identify open translation reading frames;
3. Overproduce useful RNAs and proteins; and
4. Determine the nucleotides sequences of genes and segments of DNA.
Primarily a cloning vector is necessary as a carrier of the cloned gene. Without a vector, a DNA molecule introduced into a cell would be diluted out of the cell division and eventually get lost.
Vectors are of following types:
1. PLASMIDS,
2. BACTERIOPHAGE VECTORS,
3. COSMIDS,
4. VECTORS FOT PLANT CELLS,
5. VIRUS VECTORS FOR ANIMAL CELLS,
6. SHUTTLE VECTORS,
7. MINICHROMOSOMES (YAC VECTORS AND BAC VECTORS)
8. EXPRESSION VECTORS AND
9. GENE CARTRIDGES.
THE PRESENT LECTURE WILL BE TOTALLY CENTERED AROUND THE PLASMIDS.
PLASMIDS_____________________________________________________
Plasmids are extra-chromosomal double stranded circular DNA molecules that replicate autonomously. i.e. a plasmids is a replicon (i.e. unit of genetic material capable of independent replication) that is stably inherited (maintained without specific selection) in an extrachromosomal state. Most of the plasmids are not required for the survival of the host in which they reside. But in many cases they are essential under certain conditions, such as in the presence of an antibiotic such as chloroamphenicol, ampicillin. In rhizobium leguminosarum, the agent for nitrogen fixation and module formation are located in the plasmid.
Aspects related to the plasmids:
1. Replication of plasmids: the small sized plasmids use the DNA replicative enzymes of the host cells, while the large-sized plasmids carry genes that code for the special enzymes necessary for their replication, under certain conditions the plasmids can integrate with the bacteria chromosomes. They are called episomes or integrative plasmids.
2. Size of plasmids: the size of plasmids varies from less than 1.0 kb to more than 2000 kb. Small sized plasmids are more advantageous for the gene cloning studies.
3. Plasmid copy number: the copy number is defined as-the number of molecules of a plasmid bound in a single bacterial cell. It ranges from 1 to more than 50 per cell. This is specific for a given plasmid residing in bacteria cell.
4. Amplification of the plasmids: the process of increasing the copy number is called plasmid amplification. When the bacterial culture is at its exponential phase, the antibiotic chloramphenicol is added to the medium. This arrests chromosomal DNA replication and the cell division. The culture is then incubated to another 12 hours for the plasmid molecules to replicate (since plasmid is resistant to the chloramphenicol). This step increases the number of plasmids per cell, sometimes the plasmid copy number may reach to several thousand.

5. Types of plasmids____________________________________________
Plasmids are classified into following four types:
a) Conjugative or self transmissible plasmids: these plasmids carry a set of transfer (tra) genes that enable the plasmid to transfer from one bacterium to another. Thus tra genes promotes bacterial conjugation. Generally they are of high molecular weight andare present as 1-3 copies per cell
b) Non-conjugative plasmids: these plasmids lack the tra genes. They replicate autonomously but cannot transfer to another bacterium. They generally have low molecular wright and are present in multiple copies. i.e. 20-25 copies per cell.
c) Relaxed plasmids: these plasmids are maintained as multiple copies per cell.
d) Stringent plasmids: these plasmids have a limited number of copies per cell, typically 1-2 copies per cell.
6. Isolation of plasmid DNA: the isolation of the plasmid DNA include the following steps:
a) Growth of bacteria and plasmid amplification.
The bacterial strain containing the required plasmid is grown on LB (=lauria dertani) culture medium (it contains trypton(10 g/l), yeast extract (5g/l) and sodium chloride (10 g/l). the ph of the LB medium is adjusted 7.5. late long, the cultures ae then transferred to LB medium containing proper antibiotic with small amount of chloramphenicol for 12-16 hours of incubation.
b) Breaking of bacterial cells to release their contents.
The bacterial cell are subjected to gentle cell lysis, using lysozyme and then detergent..
c) Treatment of bacterial cells extracts to remove all components except the DNA.
This is followed by the clearing of lysate by centrifugation. Centrifugation tends to sediment high molecular weight DNA (predominantly chromosomal DNA) and cell debris, leaving the small plsmid molecules and RNA in subpernatant.
d) Separation of plasmids from the chromosomal DNA.
Undamaged plasmid is comparatively compact,since plasmid is supercoiled as a consequence of having very few turns of double helix per unit length. Further purification of plasmid is done by caesium chloride density gradient ultracentrifugation of nucleic acid preparation in the presence of the ethidium bromide. Ethidium bromide tends to cause unwinding of DNA as it binds to it and simultaneously decreasing its buoyant density. Since the supercoiled plasmid DNA can unwind to only a very limited extent , it will not bind the dye and have a higher density than the other types of the DNA (which binds to dye). Because of this density difference, plasmid DNA can be separated from other types of DNA (i.e., stained DNA ) by means of isopycnic ultracentrifuge.
7. Criteria for plasmid cloning: for the selection of the suitable cloning vector the following criteria are used
a. A plasmid vector should be small and should contribute as alittle as possible to the overall size of the recombinant molecule. Low molecular weight (small sized) plasmids are easy to handle, and do not get damaged during the process of their isolation.
b. The plasmid should have the ability to confer readily selectable markers (i.e. phenotypic traits on the host cells that can be used to distinguish the transformed cell from the non-transformed ones. (in the process of transformation the naked DNA molecules are taken up by the recipient cell)
c. Plasmid should be easily propagated in the desired host so that the number of recombinant DNA molecules can be amplified.
d. The plasmid should have a large number of copies.
e. A plasmid vector should have an additional genetic marker that can be inactivated by insertion of the foreign DNA. So that the inactivated gene may help in distinguishing the cell harbouring recombinant molecules on the basis of readily altered phenotype.
Design of the future molecular vectors_________________________________
a. The vector should contain dominant genetic marker that can be expressed in a wide range of hosts (e.g. CmR is a dominant genetic marker which does not restrict the vector to particular genetic background.
b. Portioning swquences such as par or cer should be also incorporated into the vector to ensure efficient segregation of plasmids to the daughter cells.
c. Method of controlling the copy number will greatly increase the versatility of the vector.
d. Since many recombinant DNA techniques requires single strands of DNA. A vector with single stranded synthesis capability is desirable.
Refrence: genetics by p.s. verma and v. k. agarwal