Tunu C Mlambya
Tunu C Mlambya
The polymerase chain reaction (PCR) is a method of generating many copies of a specific DNA sequence. Newer technologies and sample preparation applications permit the use of PCR in a more timely manner than some conventional technologies. The amount of target nucleic acid in the sample may be extremely small, the amount of the DNA sample is limited such as in forensics and prenatal testing, or when
the quality of the DNA sample is poor, yet PCR may yield results not obtainable from other techniques. The procedure will amplify picograms of nucleic acid to micrograms in a short period of time. Because of the specificity and rapidity of the testing, PCR has just about replaced every other NA procedure in clinical labs.
The premise of PCR is based on the DNA polymerase (DNA Pol) enzyme. DNA Pol is the enzyme that incorporates nucleotides onto the end of the replicating DNA strand. Thus DNA Pol needs a DNA template, all four deoxynucleotide triphosphates (dNTPs), and free 3'OH groups or primers onto which it adds the new nucleotide in sequence on the base-pairing of dNTP with the template. DNA sequencing does
the same thing, but only uses one primer and replicates one strand versus PCR that uses two primers flanking the target sequence and replicates both strands at the same time.
First Step - DENATURE: The target DNA is denatured (melted) by heating to 295?C. The strands remain melted until the temperature is lowered and then base-pairing will reoccur.
Second Step - ANNEAL: A pair of primers is added in excess quantity to out compete the complementary strand for binding to their target region. The primers are synthetic oligonucleotides constructed to anneal to known sequences flanking either side of the target region. The temperature range for annealing can vary greatly from 40?C to 70?C.
Third Step - EXTEND: DNA Pol is added to the reaction. It mimics natural replication of DNA and copies an adjacent DNA template to extend across the area between the primers. Extension temperature is commonly 72?C and is based on the type of DNA Pol used.
CYCLE: Heat is again applied to denature DNA, followed by cooling to allow more primer to anneal and primer extension to take place. The reaction takes place in a thermocycle programmed to give rapid and accurate temperature changes. The number of cycles may vary, but most commonly 30-35 cycles are performed. For every one template, 235 copies are theoretically possible
The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is
amplified using primer mediated enzymes. DNA Polymerase synthesises new strands of DNA
complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing
3'-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3' prime
The main objective of the primer is synthesizing DNA with a free terminal end and initiation point of polymerase. A pair of primers one at the template strand while the other at the complementary strand binds on the opposite ends of the sequence being designed, likewise, the 3' corresponds to the template strand for the process of elongation.
The forward primer runs in 3'-5' while the reverse primer runs in 5'-3'. However process of elongation results in two new strands of ds DNA.
Caution: The formation of dimers occurs when the template strand lies with the complementary strand which is an unfavourable condition.
Forward and Reverse primers don't follow the complementarity rule, rather a forward primer binds to one end of one target at 5' P while the other end of 5' P occupies reverse primer. DNA primers were used most widely in PCR, unlike replication, due to:
The concept of stability in DNA primers as compared to more than RNA primers.
Since it is the unidirectional mode of ,RNA primers can not be removed after the end of the reaction.
However, the majority of the amplification is made from DNA, it is recommended to use specific nucleotide primers only.
Note: Primers participate in the replication process and have the ability to remove RNA primers by the end of the replication process while DNA primers are not.
Mutation
Are the genetic changes of DNA sequence that happen during cell division when cells make more copies of their types. Eukaryotic DNA tells the body how to form and function. Genetic mutations could lead to genetic conditions like cancer, or they could help humans better adapt to their environment over time. How does this happen? Like words in a sentence, the DNA sequence of each gene determines the amino acid sequence for the
protein it encodes.
The DNA sequence is interpreted in groups of three nucleotide bases, called codons. Each codon specifies a single amino acid in a protein
Replication and recombinant gene
REPLICATION
Replication refer to the process by which the double strand DNA molecule is copied to produce two identical DNA molecules.
DNA replication is a process in which original DNA molecule (present in nucleus of eukaryotes and cytoplasm of prokaryotes) called as parent DNA is replicated to form two new DNA molecules called as daughter DNA molecules. Parental strands act as template (guide) strand for the synthesis of daughter strands. Three different hypotheses had been proposed for the mode of DNA replication:
1. Conservative Mode: In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand. An entirely new molecule is synthesized from a DNA template and no change occurs in template (parent) DNA i.e. it remains conserved.
2. Semi-Conservative Model: In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule). It means after replication each DNA molecule has one parental strand and one newly synthesized strand.
3. 3. Dispersive Model: In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or hybrids, of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA. New molecules are made up of segments of new and old DNA.
The complete process of DNA replication can be divided into three steps:
1.Initiation
The first step occurs when DNA helicase unwinds the double helix by breaking the hydrogen bonds between the parent strands of DNA at locations called replication origins. This opens up the DNA molecule to form a Y-shape structure, which is called as replication fork.
2. Elongation
After separation of helix separated and primer synthesis DNA polymerase starts adding complementary nucleotides and synthesis of daughter strand begin. Addition of complementary nucleotides means that if A is present on parental strand then nucleotide with base T will be added on daughter strand, if G is present on daughter strand then nucleotide with base C will be added on daughter strand and vice versa. Now at this stage where
two parent strands of DNA are unwound or separated, one strand is oriented in the 5' to 3' direction and the other strand has opposite orientation in the 3' to 5' direction.
4. Termination. After elongation is complete, two new double helices have been made from the original parental DNA molecule
RECOMBINANT GENE
Recombinant of gene refer to the process by which the pieces of DNA are broken and recombined to produce new combinations of alleles. This recombination process creates genetic diversity at the level of gene that reflects differences in the DNA sequence of different organisms. In eukaryotic cells, recombination occurs during meiosis. Meiosis is a form of cell division that produces gametes or egg and sperm cells. During the
first phase of meiosis the homologous pair of maternal and paternal chromosomes align.
• During the alignment, the arms of the chromosomes can overlap and temporarily fuse causing a crossover. Crossover result in recombination and the exchange of the genetic material between the maternal and paternal chromosomes. As a result, offspring can have different combination of genes that their parents. Genes that are located further apart on the same chromosomes have a great likelihood of undergoing recombination, which
means they have a great recombination frequency.
Template
A polynucleotide that encodes the information from which another polynucleotide, of complementary sequence, is synthesized.
Strand
Refers to the single long polynucleotide chain composed of four types of nucleotides subunits. Where if two chains winds each other form double strand and the hydrogen bonds holds the chains together between the base portions of the nucleotides.
Open reading frame.
Is the portion of DNA sequence that does not include a stop codon which function as the stop signal.
Leading strand.
A single DNA strand that during DNA replication is replicated in 3’- 5’ direction simultaneously with the replication fork.
• The DNA added to the strand continuously, one complementary base at a time.
Okazaki fragment.
Are the DNA fragments or strands formed during replication. It was named Okazaki by Reiji Okazaki in 1968. Okazaki fragments are important because they are how one strand of the new DNA daughter strand is synthesized during DNA replication. To fully define Okazaki fragments we also need to understand the process of DNA replication.
Translation and transcription
Transcription
Is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA. Other segments of DNA are copied into RNA molecules called non- coding RNAs. mRNA comprises only 1% to 3% of total
RNA samples.
It involves following steps:-
1. Initiation
The RNA Polymerase binds to a sequence of DNA called the promoter, found near the beginning of a gene. Each gene has its own promoter. Once bound, RNA Polymerase separates the DNA strands, providing the single- stranded template needed for transcription.
2. Promoter clearance
RNA Polymerase must clear the promoter once the first bond has been synthesized. The promoter is a DNA sequence that signals which DNA strand is transcribed and the direction transcription proceeds.
Approximately 23 nucleotides must be synthesized before RNA Polymerase loses its tendency to slip away and prematurely release the RNA transcript.
3. Termination
Newly synthesized mRNA from the elongation complex released after cleavage of the transcript then polyadenylation process where a series of adenine residues or poly (A) tail is added to the new 3’ end of the messenger RNA strand.
Translation
Is the process that takes the information passed from DNA as messenger RNA and turns it into a series of amino acids bound together with peptide bonds. It is essentially a translation from one code ( nucleotide sequence) to another code ( amino acid sequence).
Steps involved
1. tRNA charging:
Amino acids attached to a respective tRNA as known as amino acids activation, also known as aminoacylation.
2. Initiation:
The charged tRNA attaches to the start codon (AUG), the small subunits of ribosomes binds to the mRNA and finally larger ribosomal subunit binds to create the initiation complex.
3. Elongation
According to the codons found in the mRNA, the polypeptide chain keeps growing. Each amino acid has a peptide bond attaching it to the growing chain.
Elongation continues till the whole gene is translated.
4. Termination:
After the ribosomes reaches a stop codon such as UAA, UAG, or UGA, translation is finished since these codons lack tRNAs .
When this happens the translation stops, and the newly produced polypeptide chain is released.
Concept of central dogma.
The theory stating that “ Genetic information flows only in one direction, from DNA to RNA, to Protein, or RNA directly to Protein.
It suggests that DNA contains the information needed to make all of our proteins, and that RNA is a messenger that carries this information to the ribosomes.. The ribosomes serves as factories in the cell where the information is translated from a code into the functional product.
The central dogma of biology is simply that "DNA makes RNA, makes Proteins." It is Important to note that this central dogma applies to all living organisms, including bacteria. However, some viruses are an exception. Transcription and translation is the process which allows for the synthesis of a biomolecule called proteins. In transcription, the DNA must replicate
and create ribonucleic acid or RNA.
During this process, an enzyme called RNA polymerase first attaches to a portion of DNA in the promoter region. Next, RNA polymerase adds complementary RNA nucleotides and results in the creation of a single sided strand of genetic material called messenger RNA or mRNA. In eukaryotes, the mRNA will leave the nucleus and bind to a ribosome in the cytoplasm
or rough ER to begin translation. Translation is where the actual synthesis of proteins occurs. When mRNA binds to a ribosome, it will begin to 'decode' the mRNA by reading 3 bases at a time called codons. As codons are read, transfer RNA or tRNA will arrive at the ribosome and deliver amino acids using 3 complementary bases to the mRNA, called 'anti-codons.'
From this point, a polypeptide chain of amino acids is created and will begin to grow as more amino acids arrive from tRNA. As this process continues, the amino acid chain will grow until the sequence is
complete. Once complete, the chain will disconnect from the ribosome, and fold into its complex shape depending on its function. The resulting biomolecule is the newly synthesized protein.
Enzymes used in DNA replication
Enzyme
Are remarkable molecules found in living organisms that playa crucial role in various biological processes. They act as tiny catalyst, facilitating chemical reactions in our bodies. The following enzymes involved in DNA replication.
DNA Primases, They catalyse the synthesis of short RNA molecules used as primers for DNA polymerases. Primers synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long.
DNA Ligases, Critical enzymes of DNA metabolism they catalyse (the joining of nicked DNA) is required in replication and in DNA repair pathways that require the re- synthesis of DNA.
DNA Helicases, Also essential enzyme during DNA replication which separate double- stranded DNA into single strand allowing each strand to be copied. They unwind DNA at position called origins where synthesis will be initiated.
DNA Polymerase, Is the key molecules in DNA replication since it is responsible for synthesizing DNA : they add nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template. Two strands of DNA are base paired together.
Taq Polymerase, Is the enzyme which is thermo-stable used in the amplification of DNA copies in the Polymerase Chain Reaction (PCR).
Thermo-stable DNA polymerase, eg, Taq polymerase - Thermus aquaticus (or any other thermo-stable polymerase). This bacteria normally lives in hot springs so can tolerate temperatures above 90 ⁰C.
The process begins with the identification and isolation of the gene of interest, which codes for a specific protein or has a desired function.
The gene of interest is then isolated from the organism's genomic DNA using molecular biology techniques, such as polymerase chain reaction (PCR).
The isolated gene is inserted into a vector, often a plasmid or a viral vector, which serves as a carrier for the gene.
The recombinant vector containing the gene of interest is introduced into a host organism, such as bacteria, yeast, or mammalian cells.
Gene Expression:
The host organism's cellular machinery is then used to express the recombinant gene, leading to the production of the desired protein.
1. Target amplification (e.g. polymerase chain reaction [PCR], reverse transcriptase-PCR [RT-PCR], strand displacement amplification, transcription amplification)
4. Postamplification analysis (e.g. sequencing of the amplified product, microarray analysis, and melting curve analysis, as is done in real-time PCR)
Appropriate specimen collection and storage before arrival at the molecular diagnostic laboratory are critical. Because amplification methods are so sensitive, false-positive results from trace contamination of the specimen or equipment can easily occur.