Ray wu DNA sequencing methodology understanding

Ray wu DNA sequencing methodology understanding

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I'm trying to understand the method which Ray Wu, developed to sequence the DNA 5' cohesive ends of a lamda phage. I'm reading these two papers written by him, but i stuck at the point where he uses the 32P nucleotides.

Structure and base sequence in the cohesive ends of bacteriophage lambda DNA

Nucleotide sequence analysis of DNA: II. Complete nucleotide sequence of the cohesive ends of bacteriophage λ DNA

It's not clear to me, by reading the papers, the step in which he runs the polymerization of the DNA. Did he use all the 32P nucleotides at once or each one at a time. As i understood he made the DNA pol to use the tritiated nucleotides to fill in the gaps of the 5' cohesive ends and then he cleaves with a DNAase in many oligonucleotides which later fractionize with electrophoresis. If he added all tritiated nucleotides at once how did he recognize which one is at a specific place ?

Can someone describe the process in more detail using step by step aproach?

Thank you,

DNA sequencing

Determination of precise sequence and order of DNA bases: adenine, guanine, cytosine and thymine is performed. The purpose is to analyze and compare the genetic makeup of individuals. Moreover, it has significance in evolutionary studies and species origin. No doubt, DNA sequencing technique has revolutionized the field of biotechnological research.


Recombinant DNA methods are powerful, revolutionary techniques that allow the isolation of single genes in large amounts from a pool of thousands or millions of genes and the modification of these isolated genes or their regulatory regions for reintroduction into cells for expression at the RNA or protein levels. These attributes lead to the solution of complex biological problems and the production of new and better products in the areas of medicine, agriculture, and industry.
Recombinant DNA Methodology , a volume in the Selected Methods in Enzymology series produced in benchtop format, contains a selection of key articles from Volumes 68, 100, 101, 153, 154, and 155 of Methods in Enzymology . The essential and widely used procedures provided at an affordable price will be an invaluable aid to the graduate student and the researcher.

Ray Wu, 79, a Genetic Transformer of Crops, Is Dead

Ray Wu, a biochemist and genetic engineer who helped lead research at Cornell University on genetically modifying rice and other crops to better withstand environmental stresses, died on Feb. 10 in Ithaca, N.Y. He was 79.

The cause was heart failure, his family said.

In the 1980s, Dr. Wu and other scientists at Cornell began to look at ways to make rice more resistant to insects, drought, salt water and extremes in temperature. Using genes isolated from bacteria and other sources, the researchers induced rice cells to produce certain proteins that improve the plants’ strength and resistance. For example, a gene isolated from potatoes, proteinase inhibitor II, was successfully introduced to produce a protein disruptive to the pink stem borer, an insect that can damage rice plants.

The modified plants, called transgenic rice, have since been grown in greenhouses at Cornell, in preparation for field testing in the United States and in developing countries where rice is a staple cereal crop. A collaborator of Dr. Wu’s, Ajay K. Garg, a senior research associate in molecular biology and genetics at Cornell, said the transgenic rice would eventually be crossed with rice strains native to each region, in the hope of creating healthier and higher-yielding plants.

Dr. Wu applied similar genetic influences to corn, in order to produce kernels with a higher sugar content.

The work on genetically modified plants was based in part on Dr. Wu’s earlier studies of DNA sequencing in the 1970s. In 1976, Dr. Wu and others spliced genetic material into bacteria, demonstrating that it is possible to introduce an artificial genetic message into living cells. At the time, he predicted that the procedure would one day make it possible to transplant a great range of genetic material, using cellular components known as plasmids to carry the messages.

Dr. Wu shared his findings and laboratory techniques with many other scientists through teaching and writing, leaving a “legacy in development, the training of many leading rice researchers in China, India, Korea and throughout the developing world,” said Susan R. McCouch, a professor of plant breeding and genetics at Cornell.

Ray Jui Wu was born in Peking. He received his undergraduate degree from the University of Alabama, where his father, Hsien Wu, was a biochemist. Hsien Wu collaborated in developing the Folin-Wu method of analyzing blood sugars. Ray Wu earned his doctorate in biochemistry from the University of Pennsylvania in 1955.

From 1955 to 1966, Dr. Wu conducted research at the Public Health Research Institute of the City of New York. He then joined Cornell as an associate professor of biochemistry and molecular biology, before being named a professor of biochemistry in 1972. He was chairman of biochemistry, molecular and cell biology at Cornell from 1976 to 1978.

Dr. Wu was an adviser to the Chinese and Taiwanese governments, and supported a tradition of Chinese students in the sciences at Cornell. He became an American citizen in 1961.

Ray wu DNA sequencing methodology understanding - Biology

Searching for DNA sequencing methods

In his groundbreaking 1957 presentation, Francis Crick's proposed the concept of information flow from DNA to RNA to protein, which forever changed the way of reasoning in biology. In this concept what he called the 'Central Dogma' he explained that the information flow was a one-way street and consisted of a string of nucleic acids in DNA, copied to a nucleic acid string in RNA, which in turn acts as a template for a sequence of amino acids in a protein.

This information and Watson's and Crick's structure of DNA at hand together with Robert W. Holley, Har Gobind Khorana, and Marshall W. Nirenberg solving the genetic code in the early 1960s made the importance of knowing the DNA sequence clear. However, at this time scientists didn't have adequate tools to read the order of base pairs in DNA therefore, the base sequence in any DNA was unknown at the time. It took ten or so years of intensive research work before the first DNA was sequenced. Ray Wu determined in 1971 cohesive ends of bacteriophage λ DNA (&ast) , and Frederick Sanger completed the genome of 48,502 base pairs, ten years later, using the dideoxy chain termination method in 1982 (&ast) .

In the 1960s and 1970s scientist put a lot of effort into trying to sequence DNA. Unfortunately, the protein sequencing methods did not quite work for DNA sequencing, because amino acids sequences consist of 20 different building blocks whereas DNA consists only of four and are more similar to each other, making it difficult to distinguish them. Besides, the laborious cleaving technique to sequence proteins was only possible for relatively short sequences. This technique dates back to the early 1950s when Frederick Sanger developed this Nobel Prize-winning technique and sequenced the two chains of the insulin molecule.

At that time transfer RNAs were the shortest known biologically active amino acids, around 73 to 93 nucleotides of length. Given tRNAs' short size, made it possible to use a label and cleave method analogous to Sanger's 1949 method. Robert W. Holley and coworkers used a similar technique to sequence Escherichia coli alanine tRNA, the first sequenced nucleic acid molecule, published in 1965 (&ast) .

The discovery of type II restriction enzymes in the 1970s (&ast) was the key to advance sequencing technologies. They cleave DNA near specific strings of about four to eight nucleotides long therefore, they could be used to cut DNA into small fragments that were possible to separate using electrophoresis. These enzymes cleave the double-stranded DNA so that the opposing strands have an overhang. Since the cleavage site sequence was known, this fact was an advantage in some of the early sequencing efforts, but the methods never advanced to the level where it was possible to sequence whole gene sequences.

A decisive shift started with Frederic Sanger introducing his 'plus and minus system' in 1975 (&ast) . With this approach, it was possible to determine a sequence up to about 50 base pairs in a single analysis. However, one of the deficiencies of this method was that it was difficult to measure the correct length of homopolymer stretches, such as AAA or GGG, etc. Nevertheless, Sanger used this method in 1977 to sequence the genome of bacteriophage phi X174, 5,375 nucleotides of the total estimated length of 5,400 nucleotides (&ast) .

In the same year 1977, Maxam and Gilbert published their sequencing method (&ast) , which was in many aspects similar to the Sanger's method but having an advantage of resolving homopolymer stretches.

At the end of the year 1977, Sanger published a novel sequencing concept (&ast) , based on incorporation of the chain terminating dideoxynucleotides, ddNTPs. This method could initially produce read lengths of about 100 nucleotides.

Walter Gilbert and Frederick Sanger shared the 1980 Nobel Prize in Chemistry (&ast) "for their contributions concerning the determination of base sequences in nucleic acids." together with Paul Berg "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA."

Gene sequence data analysis – Overview

The DNA sequence analysis contains various techniques and methods to identify the different features, functionalities, forms and structures of the DNA genomic elements. Several methods like sequence alignments[1], biological data searches and analytical methods[1] were used to generate the sequence analysis.

Medical Diagnosis Process for Sequencing

The ancient history of medical diagnosis process starts with the Egyptian[2] and Greece[3] civilizations. They are a perfect information resource despite the numerous information that is made available by medical researchers. This also includes DNA sequencing of the human genetic element.

A modern medical diagnosis process has been executed to find, analyze and control human health disorders. This is also entrusted to discover the treatment process, clinical improvements and diagnosis etc. The medical diagnosis process can be classified according to its own purposes and methods. The most important of these methods includes the diagnosis and screening process of health disorders. Each medical diagnosis process has its own indications and contraindications for the tests. These provide the indications to accept and reject the test diagnosis.

Challenges in Medical Diagnosis Process

There are several techniques and methodologies that are applied in different types of medical diagnosis. This diagnosis procedure is done by achieving a differential diagnosis or medical algorithms.

Medical diagnosis process is a complicated practice. This needs an appropriate clinical skill with proper expertise knowledge. These experts should be capable of analyzing and evaluating the critical clinical situations. The complex presentation in medical diagnosis process needs the interference in a probabilistic way.

In medical diagnosis a differential diagnosis may be defined as a process to distinguish the various disorders and its symptom conditions. This differential diagnosis process is used by medical experts to identify specific disease in a patient. In other ways they need to identify and eliminate all imminently life threatening conditions. Differential diagnosis has its own abbreviations.

Early DNA Sequencing Methods

The first DNA sequence was discovered in early years of 1970. This was done by various researchers through their critical research contributions which includes two dimensional chromatography[4], florescence based sequencing with DNA gene sequencer etc.

In 1970, the early identification and determination of DNA gene sequencing method based on location specific and primer extension procedure was discovered by Ray Wu at Cornell University. The present sequencing structures were classified using the classic DNA polymerase catalysis and labeling process of nucleotide. These processes were applied to categorize the lamb phage of DNA between the years 1970 to 1973.

Frederick Sanger is popular scientists who have been honored with noble prize twice to honor his findings on protein sequencing as well as sequencing of DNA. He has published and discovered a technique for sequencing DNA gene sequences through ‘chain terminating inhibitors’ by the year 1977. The various innovations in sequencing DNA genetic elements were succeeded by simultaneous advancements in recombinant technology in DNA[5]. The above advancement improves and applies in the samples of human genomes that can be easily differentiated or separated from DNA human genetic source elements than the virus elements.

Full Genomic Sequencing

The first and foremost sequencing of full DNA genome was done Bacteriophage φX17. This sequencing process was initiated in the year 1977. In 1984, the scientists of medicinal research board council(UK) have interpreted the full genomic sequencing of the virus ‘Epstein-Barr’. This virus surrounds 1,72,282 nucleotides in its gene sequence. This is considered as a great jump and a milestone in DNA sequencing.
In 1980’s, a nonradioactive technique for relocating the genetic elements of the DNA gene sequence coding was initiated. In the year 1976, the first semi-automated machine for gene sequencing was invented. In 1987 and by early 2000, DuPont’s Genesis full automated machine was invented.

HTS - High-Throughput Sequencing Methods

In current scenario, different advanced techniques in genomic research and techniques related to gene sequencing which were invented in the mid of 1990’s. There were some semi-automated and fully automated profit-making DNA sequencers by early 2000. These methods are also called as NGS. Some of the NGS methods compared are ‘Single molecule sequencing’, Pyro gene sequencing, Gene sequencing by synthesis , Ion semiconductor, combinatorial probe anchor synthesis, Nano pore Sequencing, Gene sequencing by ligation, and Chain termination.

The High-throughput Method or NGS methods are the sequencing methods which are used in genome sequencing process, RNA sequence profiling, chip- sequencing and characterization of Epi-genome. The re-sequencing is an important process because the genetic material of a particular personality type may not show every genomic discrepancy with new persons.

1. Ken Nguyen, Xuan Guo, Yi Pan (July 2016). Multiple Biological Sequence Alignment: Scoring Functions, Algorithms and Evaluation, ISBN: 978-1-118-22904-0
2. Ancient Egyptian medicine-
3. Ancient Greek medicine
4.Levitt M (May 2001). "The Birth of Computational Structural Biology". Nature Structural & Molecular Biology. 8 (5): 392–3. Doi:10.1038/87545. PMID 11323711.
5.Alipanahi, B Delong, A Weirauch, Mt Frey, Bj (Aug 2015). "Predicting The Sequence Specificities of DNA- and RNA-Binding Proteins By Deep Learning". Nat Biotechnology. 33 (8): 831–8. Doi:10.1038/Nbt.3300. PMID 26213851.

Dr. Vijay Arputharaj is a Lecturer I of Computer and Information Science in Skyline University Nigeria. He has a PhD. in Computer Science, from Bharathiar University, Coimbatore, India.

You can join the conversation on facebook @SkylineUniversityNG and on twitter @SkylineUNigeria

There Is A Whole New Universe, Waiting To Be Explored - Welcome To The DNA Universe

On 20th July, 1969, our world changed when humans walked on the surface of the moon for the first time. This pioneering achievement by Neil A. Armstrong, Buzz Aldrin, Michael Collins and all the engineers and teams of the Apollo 11 mission broadened our reality .

In 1977, Frederick Sanger and his colleagues Nicklen and Coulson introduced the chain-terminator method or dideoxy sequencing or simply Sanger sequencing as we know it. Similar to the moon landing, Sanger sequencing changed the world of biology and dominate the sequencing world for the next 30 years. Just like Armstrong, Aldrin and Collins stood on the shoulders of giants such as Hans Lippershey and Galileo Galilei who invented the telescope in 1608/1609, or the Russian cosmonaut Yuri Gagarin who was the first human in space (12th April 1961), Sanger and his colleagues also stood on the shoulders of giants:

  • Francis Crick, James Watson and Rosalind Franklin, who introduced the world to the double helical structure of DNA in 1953.
  • Marshal Nirenberg, who demonstrated that different combinations of DNA bases encode for specific amino acids in 1961.
  • Robert Holley and colleagues, who were the first to sequence yeast transfer RNA (tRNA) using RNAses with base specificity in 1965.
  • Ray Wu, who was first to decipher a short sequence of DNA by using a technique called primer extension in 1970.
  • Walter Fiers, who read the first ever DNA sequence of a whole gene - coding for a MS2 virus coating protein - in 1972.

Image: Principle of Sanger sequencing

Since 1977, the world of genomics experienced many profound changes, most notable with the introduction of next generation sequencing based on the sequencing-by-synthesis method. Nowadays, genomics approaches are used to answer scientific questions in research, pharma and diagnostics, agriculture and food, biotechnology, and medical science.

In the spirit of the first manned moon landing 50 years ago, Eurofins Genomics launches “The DNA Universe”. It represents the sheer endless applications for genomics. For every research question in the field of genomics, Eurofins Genomics has a solution. Whether you need Sanger sequencing, primers and probes, next generation sequencing, synthetic genes, CRISPR, or cell line authentication and testing for Mycoplasma contaminations… The DNA Universe always provides the genomic tools for scientists to be explorers. Reach for the stars – We are glad to assist you!

A factor of crucial importance in the universe is light speed. It is the universe’s measuring unit for velocity. In the universe of genomics, we sets the industry standard for speed with our Express services. We realised that our customers need more speed, so we give you:

What are you waiting for? Start your journey now and contact Eurofins Genomics’ mission control, free of charge, about our comprehensive products and services.

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Ray wu DNA sequencing methodology understanding - Biology

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Feature Papers represent the most advanced research with significant potential for high impact in the field. Feature Papers are submitted upon individual invitation or recommendation by the scientific editors and undergo peer review prior to publication.

The Feature Paper can be either an original research article, a substantial novel research study that often involves several techniques or approaches, or a comprehensive review paper with concise and precise updates on the latest progress in the field that systematically reviews the most exciting advances in scientific literature. This type of paper provides an outlook on future directions of research or possible applications.

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What Are The Classical DNA Sequencing Techniques?

Here’s a brief look at the different DNA sequencing methods that have defined the frontiers in genomics and metagenomics in the last four decades

The classical sequencing methods

1. Maxam-Gilbert Sequencing

1977 was a defining year for studies in genomics. Allan Maxam and Walter Gilbert are attributed for the publication of the first standardized DNA sequencing method that leveraged the chemical modification of DNA. The chemical modification of the bases resulted in cleavage at specific sites (nucleotides). It uses radioactive labeling at the 5′-end of the DNA molecule. By varying the concentration of the modifying agent, the teams can control the sites of cleavage, which generates a family of differently-sized DNA fragments. Visualization is easy – the gel is placed on an x-ray film that produces dark bands corresponding to radiolabeled DNA. The analysis is relatively simple, as long as the fragments are small and non-repetitive.

2. Sanger Sequencing

The chain termination method was also introduced in 1977 by Frederick Sanger et. al. It had been the most popular DNA sequencing technique until the standardization of other advanced methods. Sanger sequencing uses single-stranded DNA as the template, DNA primer, deoxynucleoside triphosphates (dNTPs), DNA polymerase, and di-deoxynucleoside triphosphates (ddNTPs) as chain terminators. The lack of radioactivity made the Sanger method more favorable than the Maxam-Gilbert method. After controlled DNA extension, there is a round of heat denaturation and separation of the strands using gel electrophoresis.

The Sanger method of DNA sequencing and analysis is straightforward and fast for short DNA sequences. Laboratories combine the standard DNA dye-terminator sequencing with high-throughput automated DNA sequence analyzers for the quick determination of DNA sequence.

What Are The Challenges Of Classical DNA Sequencing And Analysis Techniques?

  • The initial lag period for primer binding in the Sanger method corresponds to poor quality of DNA sequencing in the first 15 to 40 bases.
  • The efficiency of the process decreases after 700 to 900 bases.
  • The power of resolution is insufficient for larger DNA sequences.

What Is Next-Generation Sequencing

Next-generation sequencing or NGS does not refer to a single method of DNA sequencing. There are several standardized methods of DNA sequencing and analysis that fall under the NGS category. Almost all of them share a few features. They are

  1. Fast
  2. Low-cost
  3. Capable of sequencing medium to large DNA fragments
  4. High-reliability
  5. Capable of running massively parallel sequencing reactions simultaneously

All NGS methods are high-throughput techniques.

Ray wu DNA sequencing methodology understanding - Biology

First Generation Sequencing

A round 1953, Watson and Crick developed the first ever model of the DNA, giving the world inherent information of the most valuable asset to their unique identities. They took crystallographic data from Rosalind Franklin and Maurice Wilkins which alongside giving them a framework for the DNA, gave them information about the replication process and how proteins are coded. However, due to DNA&rsquos infinite length and the small number of nucleotide pairs, it became hard to configure the sequences properly.

Since DNA was a rather large piece to use as a mechanism for finding the order of the bases, scientists opted for ribosomal RNA or transfer RNA in microbials or used RNA from bacteriophages which were all much shorter than a typical eukaryotic cell and did not have the complementary strand like DNA. However, things began to slow down when instead of figuring out the actual order of the base pairs, scientists were only able to figure out the composition which marked the entry of Robert Holley in 1965. Holley and his colleagues used alanine tRNA from Saccharomyces cerevisiae to develop a nucleic acid sequence. Alongside Holley, Fred Sanger, a British biochemist used DNA fractionation to add to the existing ribosomal and transfer RNA. Fractionation occurs through the use of a pulsed field electrophoresis with alternating electric fields to separate DNA fragments according to characteristics such as their mass. Around this time, scientists broadened the scope of their research and began working with purified bacteriophages which was made possible with the emerging research in the field of genomics. Since these organisms have five hanging cohesive ends, Ray Wu and Dale Kaiser used polymerase to create radioactive nucleotides at the end in order to deduce the sequence of DNA. Despite these attempts, the bases the scientists found turned out to be shorter than usual thus calling for more alterations.

In order to reach better results, new developments had to be made using DNA fractionation. Since this process involved both gel electrophoresis and chromatography, a two way technique using the protocols of Alan Coulson and Sanger&rsquos plus and minus systems, and Allan Maxam and Walter Gilbert&rsquos cleavage techniques were adopted. A primer is used to synthesize DNA with radiolabelled nucleotides which is ended with a plus reaction, or polymerization to create extensions that end with a single type of nucleotide. The minus test is where the other three nucleotides come into play and are sequenced until there is a gap in the sequence where the fourth nucleotide from the plus reaction is absent. Afterwards, gel electrophoresis is used to analyze where each of the nucleotides are at. Using this method, Sanger and his colleagues were able to find the genome of a bacteriophage. Despite their significant similarities, the Gilbert and Maxam approach differed in how the DNA fragments were cleaved. Instead of using polymerization, a chemical substance was used to break the nucleotides up into smaller pieces. This method was more prominent than Sanger&rsquos method and is known to be the first true application of DNA sequencing.

In 1977, Sanger made another advancement in the field of DNA technology which drastically altered the manner in which sequencing was done prior to the discovery. Called the dideoxy technique, this method took advantage of the absence of a 3&rsquo hydroxyl group which are needed for the extension of DNA chains thus connecting to 5&rsquo phosphate groups. Using radiolabelled nucleotides at unequal concentrations with the DNA concentration, this slows down the process of DNA synthesis. This process is performed using DNA polymerase where DNA synthesis is performed. While doing so, modified pieces of nucleotides (dideoxynucleotides) of bases A, C, G, and T are added and since they lack a hydroxyl group, it stops the synthesis process midway. Since there are four bases, each reaction which involves the production of a DNA strand have to be given the four nucleotides separately. This process results in several different lengths of DNA fragments, each unique to its own. The next step is to denature the DNA fragment and to run it through gel electrophoresis. The simplicity and ease with which one could perform this process made it the universal method of DNA sequencing which came to be know as chain termination or simply, Sanger Sequencing.

The next few years followed up with improved techniques in this field, which was being able to incorporate more than one base in a single reaction as well as producing new machines which could identify sequences of complex species around the world. More methods that are readily used today began to be developed and resulted in what we know today as the polymerase chain reaction (PCR) and recombinant DNA technologies.

The picture at the right shows the process of a PCR technique: Polymerase Chain Reaction.

Watch the video: Sanger DNA Sequencing - Gel Electrophoresis Animation (August 2022).