Information

How were the first primers made?


I keep reading about how primers are useful in pcr -- they allow you to select a specific dna region. Similarly, in NGS or Sanger sequencing they give you a starting point. The primers I see are about 20-30 bases long.

However, how were the first primers developed? Somewhere, someone needed to come up with a 20-base sequence that bound to a specific part. Aren't there 4^20 such combinations? That's a lot of potential options to test.

How was that done?


The MIT synthetic chemist Gobind Khorana won the 1968 Nobel Prize in Chemistry for his work which successfully was able to make chains of Ribonucleic acids. The chemistry was difficult at the time but he won the prize for making specific sequences of RNA bases which were then fed to cells, resulting in specific amino acid chains, which ultimately deciphered the specific base to amino acid correspondence of the genetic code.

Khorana was only one of the chemists who worked on nucelotide synthesis, but the technique overall is simple and since nucleic acid polymers are only unbranched polymers, any sequence combination is easily obtained. Each sequential reaction adds one base. Repeat the reaction as many times as needed.

The chemistry Khorana devised was refined and easily extended to DNA polymers since the linkages are identical, the difference between DNA and RNA is only a single alcohol (-OH) group, which is not directly involved in the bond between the nucleotide bases.

By 1983 when Kary Mullis concieved the idea of PCR, short sequences of DNA and RNA could be synthesized on commercially available machines. In fact Mullis was responsible for just such a machine at Cetus Pharmaceuticals and had been trying to find a way to increase demand for his services.

The PCR oligos were the easy part of the operation. To prove the concept, the team at Cetus used a conventional DNA polymerase and add it to the PCR mix after every cycle. By 1986 the Cetus team had succeeded in performing PCR a heat tolerant DNA polymerase from Thermus aquaticus, a bacterium from a hot spring, which basically made it the technique we still use today.


The process of sequencing was, and can be, assisted by cloning a DNA fragment into a known site in a plasmid designed to help sequencing. That cloning site is flanked by a known sequence(s) that can use standard primers

In the figure "A" and "B" are just reference points on either side of the Cloning site. Of course, the plasmid extends on either side of the shown region. The plasmid is first cut open at the cloning site (upper step) and the unknown DNA fragment ligated in (middle step). Then the known 'forward' sequence can be used, with a complementary primer (shown as the arrow), to prime a sequencing reaction (lower step, dashed line). Additional modifications include (per @Chris) preparing the insert with adapters or using a plasmid with sequence that binds a 'reverse' primer (on this figure, that would be an arrow on the right hand side of "B" and pointing to the left). The reverse primer allows sequencing of the opposite strand to the forward primer and gives access to the 3' end of the inserted unknown sequence. In the early days of Sanger sequencing read lengths were quite short and often were unable to run the length of a larger insert.

Sequencing plasmids are designed so that their forward and reverse primer binding sites are unlikely to contain any sequences that may be found in the insert. Because these known primers can be used in this manner to sequence almost any insert, they are often called Universal Primers (per @AlanBoyd).


Frederick Sanger

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Frederick Sanger, (born August 13, 1918, Rendcombe, Gloucestershire, England—died November 19, 2013, Cambridge), English biochemist who was twice the recipient of the Nobel Prize for Chemistry. He was awarded the prize in 1958 for his determination of the structure of the insulin molecule. He shared the prize (with Paul Berg and Walter Gilbert) in 1980 for his determination of base sequences in nucleic acids. Sanger was the fourth two-time recipient of the Nobel Prize.


Initiation

Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. In some eukaryotes, like yeast, these locations are defined by having a specific sequence of basepairs to which the replication initiation proteins bind. In other eukaryotes, like humans, there does not appear to be a consensus sequence for their origins of replication. Instead, the replication initiation proteins might identify and bind to specific modifications to the nucleosomes in the origin region.

Certain proteins recognize and bind to the origin of replication and then allow the other proteins necessary for DNA replication to bind the same region. The first proteins to bind the DNA are said to &ldquorecruit&rdquo the other proteins. Two copies of an enzyme called helicase are among the proteins recruited to the origin. Each helicase unwinds and separates the DNA helix into single-stranded DNA. As the DNA opens up, Y-shaped structures called replication forks are formed. Because two helicases bind, two replication forks are formed at the origin of replication these are extended in both directions as replication proceeds creating a replication bubble. There are multiple origins of replication on the eukaryotic chromosome which allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.

Figure (PageIndex<1>): Replication Fork Formation: A replication fork is formed by the opening of the origin of replication helicase separates the DNA strands. An RNA primer is synthesized by primase and is elongated by the DNA polymerase. On the leading strand, only a single RNA primer is needed, and DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches, each of which must start with its own RNA primer. The DNA fragments are joined by DNA ligase (not shown).


Telomere Replication

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 9.11) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Figure 9.11 The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure 9.12) received the Nobel Prize for Medicine and Physiology in 2009.

Figure 9.12 Elizabeth Blackburn, 2009 Nobel Laureate, was the scientist who discovered how telomerase works. (credit: U.S. Embassy, Stockholm, Sweden)

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.


Double stranded DNA templates denature at a temperature that is determined in part by their G+C content. The higher the proportion of G+C, the higher the temperature required to separate the strands of template DNA. The longer the DNA molecules, the greater the time required at the chosen denaturation temperature to separate the two strands completely. If the temperature for denaturation is too low or if the time is too short at AT rich regions of the template DNA will be denatured. When the temperature is reduced later in the PCR cycle, the template DNA will reanneal into fully native condition. In PCRs catalyzed by Taq polymerase, denaturation is carried out at 94-95 o C, which is the highest temperature that the enzyme can endure for 30 or more cycles without sustaining excessive damage. In the first cycle of PCR, denaturation is sometimes carried out for 5 minutes to increase the probability that long molecules of template DNA are fully denatured. However this extended period of denaturation temperature is unnecessary for linear DNA molecules as it may be deleterious sometimes. Denaturation for 45 seconds at 94-95oC is routinely used to amplify linear DNA molecules whose GC content is <55% and higher temperature for template and/or target DNAs whose GC content is >55%. So much more heat tolerant polymerases are preferred in such cases.

The temperature used for the annealing step is critical. If the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all to the template and the yield of amplified DNA is very low. If the annealing temperature is too low, non specific annealing of primers may occur, resulting in the amplification of unwanted segments of DNA. Annealing is usually carried out 3-5o C lower than the calculated melting temperature at which the oligonucleotide primers dissociate from their templates. Many formulas exist to determine the theoretical Tm, but none of them are accurate for oligonucleotide primers for all lengths and sequences. It is best to optimize the annealing conditions by performing a series of trial PCRs at temperatures ranging from 2C to 10 C below the lower of melting temperatures calculated for the two oligonucleotide primers. Alternatively, the thermal cycler can be programmed to use progressively lower annealing temperatures in consecutive pairs of cycles ("touchdown" PCR. Instead of surveying a variety of annealing conditions in separate PCRs, optimization is achieved by exposing a single PCR to a sequential series of annealing temperatures in successive cycles of the reaction.

Extension of oligonucleotide primers is carried out at or near the optimal temperature for DNA synthesis catalyzed by the thermostable polymerase, which in the case of Taq polymerase is 72-78 o C. In the first two cycles, extension from one primer proceeds beyond the sequence complementary to the binding site of the other primer. In the next cycle, the first molecules are produced whose length is equal to the segment of DNA delimited by the binding sites of the primers. From the third cycle onwards, this segment of DNA is amplified geometrically, whereas longer amplification products accumulate arithmetically. The polymerization rate of Taq polymerase is

2000 nucleotides /minute at the optimal temperature (72-78 o C) and as a rule of thumb, extension is carried out for 1 minute for every 1000bp of product. For the last cycle of PCR, many investigators use an extension time that is 3 times longer than the previous cycles, ostensibly to allow completion of all amplified products.


The Human Genome Project

The Human Genome Project (HGP) was one of the great feats of exploration in history. Rather than an outward exploration of the planet or the cosmos, the HGP was an inward voyage of discovery led by an international team of researchers looking to sequence and map all of the genes -- together known as the genome -- of members of our species, Homo sapiens. Beginning on October 1, 1990 and completed in April 2003, the HGP gave us the ability, for the first time, to read nature's complete genetic blueprint for building a human being.

The Human Genome Project was the international research effort to determine the DNA sequence of the entire human genome.

In 2003, an accurate and complete human genome sequence was finished two years ahead of schedule and at a cost less than the original estimated budget.

Key moments and press releases from the history of the Human Genome Project.

February 15, 2021 marks the 20-year anniversary of publications reporting the draft human genome sequence.

Video testimonials from prominent members of the genomics community commemorating and celebrating the 30th anniversary of the launch of the Human Genome Project.

Explore frequently asked questions and answers about the Human Genome Project and its impact on the field of genomics.


Mastering Biology Chp. 14 HW

Use the table to sort the following ten codons into one of the three bins, according to whether they code for a start codon, an in-sequence amino acid, or a stop codon.

During translation, nucleotide base triplets (codons) in mRNA are read in sequence in the 5' → 3' direction along the mRNA. Amino acids are specified by the string of codons. What amino acid sequence does the following mRNA nucleotide sequence specify?
5′−AUGGCAAGAAAA−3′

[Before a molecule of mRNA can be translated into a protein on the ribosome, the mRNA must first be transcribed from a sequence of DNA.
The diagram shows a scheme of translation of DNA into a protein on the ribosome. There is a fragment of DNA with two strands (the strand from 3 prime to 5 prime is a template strand). The template strand is transcribed into mRNA (complementary nitrogenous bases are selected - U for A, A for T, C for G and G for C). Then the mRNA is translated into the protein of the ribosome, and each codon is translated into a certain amino acid (e. g., UGG is translated into Trp). The sequence of amino acids forms the protein.]

What amino acid sequence does the following DNA nucleotide sequence specify?
3′−TACAGAACGGTA−5′

Suppose that a portion of double-stranded DNA in the middle of a large gene is being transcribed by an RNA polymerase. As the polymerase moves through the sequence of six bases shown in the diagram below, what is the corresponding sequence of bases in the RNA that is produced?

[Diagram of DNA showing a coding strand and a template strand. Coding strand from the 3 prime end to the 5 prime end reads C C G A G T. Template strand from the 5 prime end to the 3 prime end reads G G C T C A.]

During transcription in eukaryotes, a type of RNA polymerase called RNA polymerase II moves along the template strand of the DNA in the 3'→5' direction. However, for any given gene, either strand of the double-stranded DNA may function as the template strand.

After transcription begins, several steps must be completed before the fully processed mRNA is ready to be used as a template for protein synthesis on the ribosomes.

[Once RNA polymerase II is bound to the promoter region of a gene, transcription of the template strand begins. As transcription proceeds, three key steps occur on the RNA transcript:
-Early in transcription, when the growing transcript is about 20 to 40 nucleotides long, a modified guanine nucleotide is added to the 5' end of the transcript, creating a 5' cap.
-Introns are spliced out of the RNA transcript by spliceosomes, and the exons are joined together, producing a continuous coding region.
-A poly-A tail (between 50 and 250 adenine nucleotides) is added to the 3' end of the RNA transcript.

Match the words in the left-hand column with the appropriate blank in the sentences in the right-hand column.

In eukaryotic cells, the processes of protein synthesis occur in different cellular locations.

RNA plays important roles in many cellular processes, particularly those associated with protein synthesis: transcription, RNA processing, and translation.

[In eukaryotes, pre-mRNA is produced by the direct transcription of the DNA sequence of a gene into a sequence of RNA nucleotides. Before this RNA transcript can be used as a template for protein synthesis, it is processed by modification of both the 5' and 3' ends. In addition, introns are removed from the pre-mRNA by a splicing process that is catalyzed by snRNAs (small nuclear RNAs) complexed with proteins.
The product of RNA processing, mRNA (messenger RNA), exits the nucleus. Outside the nucleus, the mRNA serves as a template for protein synthesis on the ribosomes, which consist of catalytic rRNA (ribosomal RNA) molecules bound to ribosomal proteins. During translation, tRNA (transfer RNA) molecules match a sequence of three nucleotides in the mRNA to a specific amino acid, which is added to the growing polypeptide chain.

Life as we know it depends on the genetic code: a set of codons, each made up of three bases in a DNA sequence and corresponding mRNA sequence, that specifies which of the 20 amino acids will be added to the protein during translation.

Imagine that a prokaryote-like organism has been discovered in the polar ice on Mars. Interestingly, these Martian organisms use the same DNA → RNA → protein system as life on Earth, except that
-there are only 2 bases (A and T) in the Martian DNA, and
-there are only 17 amino acids found in Martian proteins.


Arabidopsis Genetics

Norms and nomenclature

A. thaliana is often indicated simply by the genus name Arabidopsis, even though other species within the genus also are subjects of investigation. Some authors consider Arabidopsis to be a common name, printing the nonitalicized word with or without capitalization. Other English names for the plant, including Thale cress and mouse-ear cress, are rarely used by researchers.

Arabidopsis genes newly identified through mutant analysis are named for the mutant phenotype (Meinke and Koornneef 1997), whereas those identified via reverse genetics are often named for the encoded protein. Gene names are typically abbreviated to three letters. When a gene that has already been described is rediscovered in a new experiment, the previously published name is often used to avoid creating long lists of synonymous gene names. Genes and genotypes are italicized protein names are not. Wild-type names are written using capital letters mutant names are lowercase. A locus number follows the letters to distinguish different genes that can mutate to a given phenotype (for genes identified by mutation) or various homologs (for genes identified by homology), and various alleles of the same gene are enumerated following a hyphen. For example, the TRANSPORT INHIBITOR RESPONSE1 (TIR1) gene encodes the TIR1 protein (Ruegger et al. 1998). In this example, the numeral one denotes the first mutant isolated in the transport inhibitor response mutant screen. The tir1-1 protein contains a glycine-to-aspartate change caused by the tir1-1 missense mutation in the tir1-1 mutant, whereas the tir1-9 mutant harbors a T-DNA insertion in the TIR1 gene (Ruegger et al. 1998).

Following the completion of the Arabidopsis sequencing project (Arabidopsis Genome Initiative 2000), genes also have standardized names assigned by TAIR. The standard gene name includes At for A. thaliana, the nuclear chromosome number (or C for chloroplast or M for mitochondrion), and the letter g for gene followed by a unique, five-digit numerical identifier that reflects the chromosomal position. In this system, the TIR1 gene is At3g62980, indicating that the gene is on chromosome 3 with the large number reflecting a position near the bottom of the chromosome. The original annotators spaced the numbers 10 digits apart, leaving room for discovery of genes overlooked in the first annotation.

Independently collected Arabidopsis lineages are known as accessions. Arabidopsis “accessions” are groupings within the species analogous to “breeds” within animal species or “varieties” of crop plants. The differences between accessions range from easily distinguished ecotypes to nearly identical plants that were independently collected and named. The most commonly used wild type is Columbia-0 (Col-0) Landsberg erecta (Ler) and Wassilewskija (Ws) are also commonly studied. Although a number of different accessions of Col-0 may have been used for generating the reference Arabidopsis genome sequence, the Col-0 accession CS70000 has been proposed by TAIR as the reference stock (Huala et al. 2001).

Plant care and growth conditions

Seeds can be germinated directly on the surface of moistened soil. To distribute seeds more evenly when sowing, they can be suspended in a 0.1% (w/v) agar solution and distributed volumetrically using a pipette. It is not necessary to bury the seeds. However, seeds on the soil surface are susceptible to desiccation, and plastic domes or tented plastic wrap can be used to reduce evaporation for the first week or longer. If atmospheric humidity is low, plants may be damaged by sudden removal of the cover, and partially removing the plastic dome or cutting slits in plastic wrap a few days before entirely removing the cover aids survival.

For more carefully controlled experiments, such as those using specific additives or investigating aspects of root development, seedlings can be germinated and grown on sterile media in Petri dishes. For this purpose, seeds are first surface sterilized (gently enough not to kill the embryo) using bleach and detergent (Haughn and Somerville 1986), ethanol (Nelson et al. 2009), or other disinfectants. Two types of media are commonly used: (MS) medium (Murashige and Skoog 1962) and plant nutrient (PN) medium (Haughn and Somerville 1986). MS offers the convenience of commercial, premeasured media packets. PN medium is less convenient, requiring preparation of several stock solutions and mixing of these stocks for each batch of medium (Haughn and Somerville 1986), but offers more user control over the composition of the growth medium. Media may be supplemented with sucrose to promote even germination and to allow the early development of certain metabolic mutants (Pinfield-Wells et al. 2005). Even mutants that require supplemented growth medium for germination can often survive transfer to soil once established (Zolman et al. 2000). Transfer to soil is generally required for a robust seed set.

It is possible to grow Arabidopsis in soil or aseptically on plates in ambient air under common lights, including LED, fluorescent, or incandescent bulbs. Lighted plant growth chambers allow precise control of day length and circulate air to maintain stable, user-defined temperatures while limiting condensation in closed Petri dishes. For experiments testing responses to light or to protect photosensitive chemicals, LED-equipped growth chambers offer fine control of light wavelength and intensity. Alternatively, white light can be filtered through colored plastic sheets (Stasinopoulos and Hangarter 1990), or plates can be wrapped in foil to provide darkness. To test plant responses to other environmental parameters, some incubators can regulate humidity and atmospheric gases such as carbon dioxide.

Arabidopsis plants generally self-pollinate, but the small flowers can be manually crossed with some practice. The ovules of a flower are receptive before the pollen is mature. Therefore, the sepals, petals, and anthers are removed from a recipient (female) unopened flower bud with forceps and then anthers from a mature (open) donor flower are used to dust the exposed stigmatic papillae with pollen. F1 seeds are ready for harvest in ∼2 weeks.


Processing of tRNAs and rRNAs

rRNA and tRNA are structural molecules that aid in protein synthesis but are not themselves translated into protein.

Learning Objectives

Describe how pre-rRNAs and pre-tRNAs are processed into mature rRNAs and tRNAs.

Key Takeaways

Key Points

  • Ribosomal RNA (rRNA) is a structural molecule that makes up over half of the mass of a ribosome and aids in protein synthesis.
  • Transfer RNA (tRNA) recognizes a codon on mRNA and brings the appropriate amino acid to that site.
  • rRNAs are processed from larger pre-rRNAs by trimming the larger rRNAs down and methylating some of the nucleotides.
  • tRNAs are processed from pre-tRNAs by trimming both ends of the pre-tRNA, adding a CCA trinucleotide to the 3′ end, if needed, removing any introns present, and chemically modified 12 nucleotides on average per tRNA.

Key Terms

  • anticodon: a sequence of three nucleotides in transfer RNA that binds to the complementary triplet (codon) in messenger RNA, specifying an amino acid during protein synthesis

Processing of tRNAs and rRNAs

The tRNAs and rRNAs are structural molecules that have roles in protein synthesis however, these RNAs are not themselves translated. In eukaryotes, pre-rRNAs are transcribed, processed, and assembled into ribosomes in the nucleolus, while pre-tRNAs are transcribed and processed in the nucleus and then released into the cytoplasm where they are linked to free amino acids for protein synthesis.

Ribosomal RNA (rRNA)

The four rRNAs in eukaryotes are first transcribed as two long precursor molecules. One contains just the pre-rRNA that will be processed into the 5S rRNA the other spans the 28S, 5.8S, and 18S rRNAs. Enzymes then cleave the precursors into subunits corresponding to each rRNA. In bacteria, there are only three rRNAs and all are transcribed in one long precursor molecule that is cleaved into the individual rRNAs. Some of the bases of pre-rRNAs are methylated for added stability. Mature rRNAs make up 50-60% of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities.

The eukaryotic ribosome is composed of two subunits: a large subunit (60S) and a small subunit (40S). The 60S subunit is composed of the 28S rRNA, 5.8S rRNA, 5S rRNA, and 50 proteins. The 40S subunit is composed of the 18S rRNA and 33 proteins. The bacterial ribosome is composed of two similar subunits, with slightly different components. The bacterial large subunit is called the 50S subunit and is composed of the 23S rRNA, 5S rRNA, and 31 proteins, while the bacterial small subunit is called the 30S subunit and is composed of the 16S rRNA and 21 proteins.

The two subunits join to constitute a functioning ribosome that is capable of creating proteins.

Transfer RNA (tRNA)

Each different tRNA binds to a specific amino acid and transfers it to the ribosome. Mature tRNAs take on a three-dimensional structure through intramolecular basepairing to position the amino acid binding site at one end and the anticodon in an unbasepaired loop of nucleotides at the other end. The anticodon is a three-nucleotide sequence, unique to each different tRNA, that interacts with a messenger RNA (mRNA) codon through complementary base pairing.

There are different tRNAs for the 21 different amino acids. Most amino acids can be carried by more than one tRNA.

Structure of tRNA: This is a space-filling model of a tRNA molecule that adds the amino acid phenylalanine to a growing polypeptide chain. The anticodon AAG binds the codon UUC on the mRNA. The amino acid phenylalanine is attached to the other end of the tRNA.

In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple processing steps before the mature tRNA is ready for use in translation. In bacteria, multiple tRNAs are often transcribed as a single RNA. The first step in their processing is the digestion of the RNA to release individual pre-tRNAs. In archaea and eukaryotes, each pre-tRNA is transcribed as a separate transcript.

The processing to convert the pre-tRNA to a mature tRNA involves five steps.

1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.

2. The 3′ end of the pre-tRNA is cleaved off.

3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides is added to the 3′ end of the pre-tRNA after the original 3′ end is trimmed off. Some bacteria and archaea pre-tRNAs already have the CCA encoded in their transcript immediately upstream of the 3′ cleavage site, so they don’t need to add one. The CCA at the 3′ end of the mature tRNA will be the site at which the tRNA’s amino acid will be added.

4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On average about 12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine (ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D). But over 100 other modifications can occur.

5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced out. Introns are rarer in bacterial pre-tRNAs, but do occur occasionally and are spliced out.

After processing, the mature pre-tRNA is ready to have its cognate amino acid attached. The cognate amino acid for a tRNA is the one specified by its anticodon. Attaching this amino acid is called charging the tRNA. In eukaryotes, the mature tRNA is generated in the nucleus, and then exported to the cytoplasm for charging.

Processing of a pre-tRNA.: A typical pre-tRNA undergoing processing steps to generate a mature tRNA ready to have its cognate amino acid attached. Nucleotides that are cleaved away are shown in green. Chemically-modified nucleotides are in yellow, as is the CAA trinucleotide that is added to the 3′ end of the pre-tRNA during processing. The anticodon nucleotides are shown in a lighter shade of red.


  • In order to perform PCR, you must know at least a portion of the sequence of the DNA molecule that you wish to replicate.
  • You must then synthesize primers: short oligonucleotides (containing about two dozen nucleotides) that are precisely complementary to the sequence at the 3' end of each strand of the DNA you wish to amplify.
  • The DNA sample is heated to separate its strands and mixed with the primers.
  • If the primers find their complementary sequences in the DNA, they bind to them.
  • Synthesis begins (as always 5' -> 3') using the original strand as the template.
  • The reaction mixture must contain
    • all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
    • a DNA polymerase. It helps to use a DNA polymerase that is not denatured by the high temperature needed to separate the DNA strands.

    Using automated equipment, each cycle of replication can be completed in less than 5 minutes. After 30 cycles, what began as a single molecule of DNA has been amplified into more than a billion copies (2 30 = 1.02 x 10 9 ).

    With PCR, it is routinely possible to amplify enough DNA from a single hair follicle for DNA typing. Some workers have successfully amplified DNA from a single sperm cell. The PCR technique has even made it possible to analyze DNA from microscope slides of tissue preserved years before. However, the great sensitivity of PCR makes contamination by extraneous DNA a constant problem.

    External Link
    View an animation of the PCR.
    Please let me know by e-mail if you find a broken link in my pages.)


    Watch the video: Homemade Primer 2020 Edition (January 2022).