MODIFICATION OF PCR

Some modification of PCR

PCR RFLP PCR RAPD RT PCR NESTED PCR MULTIPLEX PCR QUANTITATIVE PCR

REVERSE TRANSCRIPTION PCR (RT PCR)

RT-PCR is technique f amplifying a d fi d RT PCR i a t h i for lif i defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA (cDNA), followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step p p process. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction which is also marketed as RT PCR l k d RT-PCR.

RT PCR

The fi t t Th first step of RT-PCR, i called th "fi t strand f RT PCR is ll d the "first t d reaction" Complementary DNA (cDNA) is made from a messenger RNA template using dNTPs and an RNA dependent RNA-dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription. The above components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 37C or 30 min at 42C

RT PCR

After the reverse transcriptase reaction is complete and complete, complementary DNA has been generated, standard polymerase chain reaction, termed the "second strand reaction," is initiated. A thermostable DNA polymerase and the upstream and downstream DNA primers are added. The reaction is heated to temperatures above 37C to facilitate sequence specific binding of DNA primers to the cDNA Further heating allow the thermostable DNA polymerase to make double-stranded DNA from the primer bound cDNA. The reaction is heated to approximately 95C to separate the two 95 C DNA strands The reaction is cooled enabling the primers to bind again and the y p cycle repeats. After approximately 30 cycles, millions of copies of the sequence of interest are generated. The original RNA template is degraded by RNase H, leaving pure cDNA (plus spare primers).

mRNA 5 RNA

AAAAA TTTTT Reverse transcriptase + dNTPs AAAAA TTTTT Rnase H AAAAA TTTTT DNA polymerase + dNTP dNTPs DNA ligase AAAAA TTTTT

3 Oligo(dT) primer

First strand cDNA synthesis

mRNA 5 cDNA 3

3 5

Nicking step

Second strand cDNA synthesis ds DNA cDNA

RT PCR

This process can be simplified into a single step process by the use of wax beads containing the required enzymes for the second stage of the p g process which are melted, releasing their contents, on heating for primer annealing in the second strand reaction.

Usage of RT-PCR RTThe exponential amplification via re erse e ponential ia reverse transcription polymerase chain reaction provides for a highly sensitive technique, where a very low copy g y q y py number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and quantitatively in the determination of and, quantitatively, the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. i In plant virology, RT-PCR is commonly used for diagnosis and study of RNA viruses viruses.

Nested PCR
Nested PCR uses two sets of amplification primers. The target DNA sequence of one set of primers (termed "inner" inner primers) is located within the target sequence of the second set of primers (termed "outer" primers). In practice, a standard PCR reaction is first run with the sample using the "outer primers". Then a second PCR reaction is run with the "inner primers using the inner primers" product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by reamplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because the inner primers amplify only if the first PCR reaction yielded a specific product. lif l th fi t ti i ld d ifi d t

As is evident in the figure, the outer primers were able to detect the viral DNA , y down to a 10-5 dilution. In contrast, the full nested PCR system detected the viral DNA down to a 10-8 dilution. Thus, the nested system is approximately 1000 times more sensitive than the single primer system.

PCR for four Dengue serotypes (two step nested RT-PCR) RT PCR) 1 : Molecular Weight Marker y p by 100bp. 2 : Dengue 1 amplicon 3 : Dengue 2 amplicon 4 : Dengue 3 amplicon 5 : Dengue 4 amplicon

Quantitative PCR
Quantitative pol merase Q antitati e polymerase chain reaction (Q PCR) (Q-PCR) is a modification of polymerase chain reaction used to rapidly measure the q p y quantity of a p y product of polymerase chain reaction. It is preferably d i f bl done i real-time, in l ti It is commonly used to determine whether a genetic sequence is present, and if it is present, to determine the number of copies in the sample.

Quantitative PCR

The three commonly used methods of quantitative polymerase chain reaction :

agarose gel electrophoresis, SYBR Green, a double stranded DNA dye, fluorescent reporter probe.
The latter two of these three can be analysed in realreal time, forming real-time polymerase chain reaction.

Quantitative PCR using Agarose Gel

Quantitative PCR studies were carried out on selected clones using genespecific specially designed primers. Whole cDNA populations were used as PCR templates, and the fragment of interest was amplified in successive PCR cycles. The rate of PCR amplification in one population vs. another reflects differences in expression of the gene in the two populations

Quantitative PCR using Dye

Using U i SYBR Green dye is more accurate than the gel method, G d i t th th l th d and gives results in real time. A DNA binding dye binds all newly synthesized double stranded (ds)DNA and an increase in fluorescence intensity is measured, thus allowing initial concentrations to be determined.
The reaction (PCR) is prepared as usual, with the addition of fluorescent dsDNA dye. The reaction is run, and the levels of fluorescence are run monitored; the dye only fluoresces when bound to the dsDNA. With reference to a standard sample, the dsDNA concentration in the PCR can be determined determined.

Quantitative PCR using Fluorescent

Using fluorescent reporter probes is the most accurate and most reliable of the methods, but also the most expensive. It uses a sequence specific RNA or DNA based probe so as to only quantify the probe sequence and not all double stranded DNA DNA.

Multiplex PCR p
The use of multiple primer pairs in one amplification reaction. This technique enable to amplify several DNA target from one sample unit. In plant p p pathology, it is commonly used for gy, y detecting multiple pathogen infection.

Multiplex PCR p

.
Multiplex PCR detection of seeds of small broomrape (377 bp amplicon) and the internal control (555 bp amplicon) extracted from soil. soil DNA from soil with 0 (lanes 1 and 2), 5 (lanes 3 and 4), and 10 (lanes 5 and 6) seeds added; small broomrape positive control (lane 7); water control (lane 8); 100 bp DNA ladder (lane L).

DNA SEQUENCING Q
Maxam & Gilbert (Enzymatic) Sanger ( g (Dideoxynucleotide) y )

Will be discussed later (PH)