Amplification of Genomic DNA Fragments

OrR

Amplification

• To get particular DNA in large amount• Fragment size shouldn’t be too long• The nucleotide sequence at each end is

known.

How to get known sequence from both end of particular genome fragment?

• Generally we do partially digestion of a genome.

• Result fragments with known RE site in both ends.

• Insert those in vector/plasmid particular region.• We know the sequence of the plasmid.• Taking some part of the plasmid along with

restriction site as primers.

PCR reaction for amplification

Steps in PCR reaction:

Summary of PCR cycle:

The Power of Polymerase Chain Reaction

1. Number of Copiesexponential progression: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, and so forth, doubling with each cycle of replicationNumber of Copies

starting with a single molecule, 25 rounds of DNA replication will result in 225 = 3.4 x 107 molecules.

2. Requires Trace Amounts of Template DNAThe major advantage of PCR amplification is that it requires only trace amounts of template DNA.

amplification is usually reliable with as few as 10–100 template molecules, which makes PCR amplification 10,000–100,000 times more sensitive than detection via nucleic acid hybridization.

The Power of Polymerase Chain Reaction

3. SensitivityThe exquisite sensitivity of PCR amplification has led to its use in DNA typing for criminal cases in which a minuscule amount of biological material has been left behind by the perpetrator (skin cells on a cigarette butt or hair-root cells on a single hair can yield enough template DNA for amplification).

4. The Limitations of Polymerase Chain Reaction

(1) The principal limitation of the technique is that the DNA sequences at the ends of the region to be amplified must be known so that primer oligonucleotides can be synthesized.

(2) In addition, sequences longer than about 5,000 base pairs cannot be replicated efficiently by conventional PCR procedures, although there are modifications of PCR that allow longer fragments to be amplified. On the other hand, many applications require amplification of relatively small fragments.

6. Designing the Oligonucleotide Primers for a PCRThe primers are the key to the success or failure of a PCR experiment. If the primers are designed correctly the experiment results in amplification of a single DNA fragment, corresponding to the target region of the template molecule.

If the primers are incorrectly designed the experiment will fail, possibly because no amplification occurs, or possibly because the wrong fragment, or more than one fragment, is amplified

Each primer must, of course, be complementary (not identical) to its template strand in order for hybridization to occur, and the 3’ ends of the hybridized primers should point toward one another.

The DNA fragment to be amplified should not be greater than about 3 kb in length and ideally less than 1 kb. Fragments up to 10 kb can be amplified by standard PCR techniques, but the longer the fragment the less efficient the amplification and the more difficult it is to obtain consistent results.

Length of the primers

If the primers are too short they might hybridize to non-target sites and give undesired amplification products.

Why not simply make the primers as long as possible?

The length of the primer influences the rate at which it hybridizes to the template DNA, long primers hybridizing at a slower rate.

In practice, primers longer than 30-mer are rarely used.

Working Out the Correct Temperatures to Use

The annealing temperature is the important one because, again, this can affect the specificity of the reaction.

The ideal annealing temperature must be low enough to enable hybridization between primer and template, but high enough to prevent mismatched hybrids from forming. This temperature can be estimated by determining the melting temperature or Tm of the primer–template hybrid.

Tm = (4 x [G + C]) + (2 x [A + T])°Cin which [G + C] is the number of G and C nucleotides in the primer sequence, and[A + T] is the number of A and T nucleotides.

After the PCR: Studying PCR Products

Three techniques are particularly important:

1. Gel electrophoresis of PCR products2. Cloning of PCR products3. Sequencing of PCR products.

Gel Electrophoresis of PCR Products

A band representing the amplified DNA may be visible after staining, or if the DNA yield is low the product can be detected by Southern hybridization. If the expected band is absent, or if additional bands are present, something has gone wrong and the experiment must be repeated.

the presence of restriction sites in the amplified region of the template DNA can be determined by treating the PCR product with a restriction endonuclease before running the sample in the agarose gel (Figure 6.7). This is a type of restriction fragment length polymorphism (RFLP) analysis and is important both in the construction of genome maps and in studying genetic diseases.

Cloning PCR Products

1. A special cloning vector which carries thymidine (T) overhangs and which can therefore be ligated to a PCR product

Special vectors of this type have also been designed for use with the topoisomerase ligation method, and this is currently the most popular way of cloning PCR products.

(2) A second solution is to design primers that contain restriction sites. After PCR the products are treated with the restriction endonuclease, which cuts each molecule within the primer sequence, leaving sticky-ended fragments that can be ligated efficiently into a standard cloning vector

Conventional PCR vs. Real-Time PCR

Conventional PCR

(1)Most conventional PCR-based tests used in molecular biology laboratories needed to be performed in dedicated spaces to control or reduce contamination that was always a threat for producing false positive test results.

(2)Conventional PCR assays also require multiple manipulations including (i) initial amplification of target nucleic acid, (ii) detection of amplified product by gel electrophoresis, and (iii) then confirmation by an alternative method such as southern blotting or chemiluminescence techniques.

(3)In general, a conventional PCR assay would require a minimum of at least 4-6 hours from the time that extracted nucleic acid is placed into a thermal cycler to begin amplification to subsequent product detection.

Real-Time PCR

1. Real Time (amplification and detection simultaneously): The new instruments combine thermo cycling or target DNA amplification with the ability to detect amplified target by fluorescently labeled probes as the hybrids are formed (i.e., detection of amplicon in real time).

2. Avoidance of Cross Contamination: As both amplification and product detection can be accomplished in one reaction vessel without ever opening the major concern of cross contamination of samples with amplified product associated with conventional PCR assays is greatly lessened

3. Quantitate PCR Products: These instruments are not only able to measure amplified product (amplicon) as it is made, but because of this capability, they are also able to quantitate the amount of product and thereby determine the number of copies of target in the original specimen.

4. Reduce Time: The amount of time to complete a real-time PCR-based assays because the time required for the post-PCR detection of amplified product is eliminated by the use of fluorescent probes. Also, some systems are able to perform rapid thermal cycling based on instrument design, detecting product in as little as 20 to 30 minutes.

Real-Time PCR Enables the Amount of Starting Material to be Quantified

• The amount of product that is synthesized during a set number of cycles of a PCR depends on the number of DNA molecules that are present in the starting mixture.

• If there are only a few DNA molecules at the beginning of the PCR then relatively little product will be made, but if there are many starting molecules then the product yield will be higher.

• This relationship enables PCR to be used to quantify the number of DNA molecules present in an extract.

Carrying Out a Quantitative PCR Experiment

In quantitative PCR (qPCR) the amount of product synthesized during a test PCR is compared with the amounts synthesized during PCRs with known quantities of starting DNA.

Although easy to perform, this type of qPCR is imprecise, because large differences in the amount of starting DNA give relatively small differences in the band intensities of the resulting PCR products.

Real-time PCR

1. A dye that gives a fluorescent signal when it binds to double-stranded DNA can be included in the PCR mixture.

This method measures the total amount of double-stranded DNA in the PCR at any particular time, which may over-estimate the actual amount of the product because sometimes the primers anneal to one another in various non-specific ways, increasing the amount of double-stranded DNA that is present.

2. A short oligonucleotide called a reporter probe, which gives a fluorescent signal when it hybridizes to the PCR product, can be used.

Because the probe only hybridizes to the PCR product, this method is less prone to inaccuracies caused by primer-primer annealing.

Each probe molecule has pair of labels.

A fluorescent dye is attached to one end of the oligonucleotide, and a quenching compound, which inhibits the fluorescent signal, is attached to the other end

Real-Time PCR can also Quantify RNA

Real-time PCR is often used to quantify the amount of DNA in an extract, for example to follow the progression of a viral infection by measuring the amount of pathogen DNA that is present in a tissue

PCR Amplification of Full-Length cDNAs

Gene Synthesis by PCR