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    sequence read 3 was synthesized by sequencing which strand of dna?

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    The Order of Nucleotides in a Gene Is Revealed by DNA Sequencing

    The Order of Nucleotides in a Gene Is Revealed by DNA Sequencing

    All of the information needed to build and maintain an organism — whether it's a human, a dog, or a bacterial cell — is contained in its DNA. DNA molecules are composed of four nucleotides, and these nucleotides are linked together much like the words in a sentence. Together, all of the DNA "sentences" within a cell contain the instructions for building the proteins and other molecules that the cell needs to carry out its daily work.

    How do researchers "read" gene sequences?

    Determining the order of the nucleotides within a gene is known as DNA sequencing. The earliest DNA sequencing methods were time consuming, but a major breakthrough came in 1975 with the development of the process called Sanger sequencing. Sanger sequencing is named after English biochemist Frederick Sanger, and it is sometimes also referred to as chain-termination sequencing or dideoxy sequencing. Some 25 years after its creation, the Sanger method was used to sequence the human genome, and, with the addition of many technological improvements and modifications, it remains an important method in laboratories across the world today.

    How does Sanger sequencing work?

    Sanger sequencing is modeled after the natural process of DNA replication, and it uses dummy nucleotides to stop replication whenever a specific nucleotide is encountered. Because this truncated replication occurs over and over again, nucleic acids of varying lengths accumulate and can be used to determine the position of each nucleotide in the sequence.

    Understanding DNA replication

    Figure 1: DNA polymerase assembles nucleotides to make a new DNA strand.

    In order to understand how Sanger sequencing works, it's first necessary to understand the process of DNA replication as it exists in nature. DNA is a double-stranded, helical molecule composed of nucleotides, each of which contains a phosphate group, a sugar molecule, and a nitrogenous base. Because there are four naturally occurring nitrogenous bases, there are four different types of DNA nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). Within double-stranded DNA, the nitrogenous bases on one strand pair with complementary bases along the other strand; in particular, A always pairs with T, and C always pairs with G. Then, during DNA replication, the two strands in the double helix separate. This allows an enzyme called DNA polymerase to access each strand individually (Figure 1). As the DNA polymerase moves down the single-stranded DNA, it uses the sequence of nucleotides in that strand as a template for replication. Thus, whenever the DNA polymerase recognizes a T in the template strand, it adds an A to the complementary daughter strand it is building; similarly, whenever it encounters a C in the original strand, it adds a G to the daughter strand. This process happens along both strands simultaneously, resulting in the eventual production of two double-helical molecules, each of which contains one "old" strand and one "new" strand of DNA.

    Setting up the sequencing experiment

    The Sanger method relies upon a variation of the replication process described above in order to determine the sequence of nucleotides in a segment of DNA. Before Sanger sequencing can begin, however, researchers must first make many copies of, or amplify, the DNA segment they wish to sequence. This is done either by cloning the DNA or by triggering the polymerase chain reaction (PCR). Once the DNA has been amplified, it is heated so that the two strands separate, and a synthetic primer is added to the mixture. The primer's sequence is complementary to the first piece of target DNA, which means that the primer and the DNA target bind with each other. At this point, the target sequence is exposed to a solution that contains DNA polymerase and all of the nucleotides required for synthesis of the complementary DNA strand — along with one special ingredient.

    Adding ddNTPs

    Figure 2: The four ddNTPs.

    As described above, the next major step in the Sanger process is to expose the target sequence to DNA polymerase and significant amounts of all four nucleotides. In their unbound form, nucleotides have three phosphate groups and are formally called deoxynucleotide triphosphates, or dNTPs (where the "N" is a placeholder for A, T, G, or C). During the construction of a new DNA strand, a molecule called a hydroxyl group (which contains an oxygen atom and a hydrogen atom) attaches to the sugar of the last dNTP in the strand and chemically binds to the phosphate group on the next dNTP. This binding causes the DNA chain to grow. In Sanger sequencing, however, a special type of "dummy" nucleotide is included with the regular dNTPs that surround the growing DNA strand. These special nucleotides are known as dideoxynucleotide triphosphates, or ddNTPs (Figure 2), and they lack the crucial hydroxyl group that is attached to the sugar of dNTPs. Therefore, whenever a ddNTP is added to a growing DNA strand, it is unable to chemically bind with the next nucleotide in the chain, and the DNA strand stops growing.

    Source : www.nature.com

    DNA Sequence

    DNA Sequence

    The DNA sequences that flank the point of insertion of a transposon insertion in the chromosomes of bacteria, yeast, and other organisms can be determined rapidly by cloning and polymerase chain reaction (PCR) methods.

    From: Encyclopedia of Genetics, 2001

    Related terms:

    ChromosomeNeoplasmEpigenomicsProteinGene ExpressionDNAPolymerase Chain ReactionAllele

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    Molecular Genetic Technology

    Robert Resnik MD, in Creasy and Resnik's Maternal-Fetal Medicine: Principles and Practice, 2019

    DNA Sequencing

    Sequencing determines the complete nucleotide sequence, or specific order of nucleotides in a gene. By listing the full genetic code, variations from an accepted “normal” (reference or consensus sequence) may be discovered. This has the potential to uncover pathogenic variants as well as benign variants, and given our limited understanding of how the genome is translated, will potentially identify what are now VUS into a defined clinical phenotype.

    The original technique of sequencing, called Sanger sequencing, involves synthesizing multiple copies of DNA that is complementary to a single-stranded template of interest using nucleotide-specific chain terminators, or dideoxynucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP). This generates synthesized fragments of varying length that can be arranged by size, and the reactions containing each terminating base (A, G, C, or T) are kept separated. Then, by “reading” the terminating base of the synthesized copies from smallest to largest, the sequence of the original single-stranded template is revealed. This molecular method was revolutionary but very time consuming, labor intensive, and limited to relatively short DNA sequences.

    Next-generation sequencing (NGS) has revolutionized genetic sequencing capabilities, and moved the fields of science and medicine into the “post-genome” era. NGS involves preparation of a full DNA library (no longer one small segment at a time) by amplifying (making many copies) and fragmenting the DNA source of interest (genomic DNA, coding DNA). After amplification and fragmenting, the DNA library consists of thousands or even millions of small overlapping DNA copy fragments, which are physically bound to a solid surface (platform specific, often beads or glass slides). The fragments are loaded into specialized multiplex machines for parallel sequencing; in other words, the sequence of every fragment on the surface can be assayed simultaneously. Individual sequence reads are subsequently aligned (recall the expected overlap due to random fragmentation of many identical copies of DNA) using various bioinformatics platforms for comparison to a reference sequence. The full set of aligned reads reveals the entire sequence of the starting DNA product. Thus, NGS applies the concept of Sanger sequencing to an entire genome with results produced in matter of hours. NGS is a very thorough and powerful method of generating genetic sequence information. Whereas CMA can simultaneously assess many snippets of DNA sequence from representative regions across the genome, NGS can provide theentire sequence of the substrate DNA, in a matter of hours. This could be likened to quickly finishing a book by reading only the first and last page of every chapter (microarray) versus developing true speed-reading capability to read every single word within a matter of hours.

    Whole exome sequencing (WES), which includes only the protein-coding region of DNA, and whole genome sequencing (WGS) are currently in use mostly in the pediatric setting, and are considered the “next frontier” in prenatal genetic diagnostic techniques. WES is designed to determine the DNA sequence of the 20,000 to 25,000 genes that code for known proteins (about 1.5% of the full genome). This coding portion, or exome, is said to contain up to 85% of the variants that have been demonstrated to cause genetic disorders19; however, it is likely that many of the variants in noncoding portions of the genome also play a significant role in human disease. By comparison, WGS requires sequencing of the full genome, including noncoding regions and introns. Data generated from sequencing methods are vast. The primary limitation of both WES and WGS is that accurate clinical interpretation lags behind the ability to generate sequence data. This is because sequencing is high throughput and there are currently no similar high-throughput assays of function to assess putative pathologic sequence findings. This technology generates thousands of variants, and new databases, such as ClinVar and ClinGen as well as the Human Gene Mutation Database, allow for evolving interpretation as more sequence data become available. Curation of variants remains a problem and will be a challenge for years to come. There is enough natural variation in the human genome that any putative finding requires rigorous validation at the level of sequence, molecular function, and interaction within the full biological system. Current recommendations from ACOG and SMFM specify that the use of WES or WGS for prenatal diagnosis be limited to clinical trials until these techniques can be further validated in prenatal samples.109

    View chapter on ClinicalKey

    DNA SEQUENCING

    K.R. Mitchelson, in Encyclopedia of Analytical Science (Second Edition), 2005

    Introduction

    DNA sequencing is very big business. Approximately US$3 billion was spent in 2003 on sequencing reagents and enzymes, and on the analyzer equipment and software for automated sequence acquisition. The majority of this sequence output was determined using capillary electrophoresis (CE) technology, which has commensurately developed rapidly over the past 10 years. CE offers high resolution and high throughput, automatic operation, and data acquisition, with online detection of dyes bound to DNA extension products. Operational advances such as pulsed-field and graduated electric fields and automated thermal ramping programs as the run progresses result in higher base resolution and longer sequence reads. Advanced base-calling algorithms and DNA marker additives that utilize known fragment sizing landmarks can also help to improve fragment base-calling, increasing call accuracy and read lengths by 20–30%. Despite the high efficiency of CE sequencers, the complete delineation of the human genome and its implication for genome-wide analysis for personalized medicine is driving the development of devices and chemistries capable of massively increased sequence throughput, compared to the conventional CE sequencers. Miniaturization of CE onto chip-based devices provides all of the above facilities – a significant improvement in the speed and improved automation of analysis. New array-based sequencing devices also promise a quantum increase in efficiency. Each of these new devices provides an extremely high throughput, high-quality-data, and low-process costs. This article also examines the automation and improvement of sequencing processes, DNA amplification processes, and alternative approaches to sequencing.

    Source : www.sciencedirect.com

    LS7C Week 8 Flashcards

    Start studying LS7C Week 8. Learn vocabulary, terms, and more with flashcards, games, and other study tools.

    LS7C Week 8

    ChoiceA., A genomic sequence is broken into small fragments of a few hundred base pairs.

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    How is DNA sequencing accomplished?

    Question 1 choices

    ChoiceA., A genomic sequence is broken into small fragments of a few hundred base pairs.ChoiceB., Whole chromosomes are sequenced in one piece.ChoiceC., Each gene is separated from the others and individually sequenced.ChoiceD., The DNA is converted to RNA.

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    ChoiceA., They are analyzed in a machine that determines the base sequence.

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    How is the actual base sequence of DNA fragments determined?

    Question 2 choices

    ChoiceA., They are analyzed in a machine that determines the base sequence.ChoiceB., They are analyzed in a powerful microscope.ChoiceC., They are translated into proteins and the amino acid sequence is determined.ChoiceD., They are carefully lined up with known sequences from other organisms.

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    1/76 Created by mr123josh

    Terms in this set (76)

    ChoiceA., A genomic sequence is broken into small fragments of a few hundred base pairs.

    How is DNA sequencing accomplished?

    Question 1 choices

    ChoiceA., A genomic sequence is broken into small fragments of a few hundred base pairs.ChoiceB., Whole chromosomes are sequenced in one piece.ChoiceC., Each gene is separated from the others and individually sequenced.ChoiceD., The DNA is converted to RNA.ChoiceA., They are analyzed in a machine that determines the base sequence.

    How is the actual base sequence of DNA fragments determined?

    Question 2 choices

    ChoiceA., They are analyzed in a machine that determines the base sequence.ChoiceB., They are analyzed in a powerful microscope.ChoiceC., They are translated into proteins and the amino acid sequence is determined.ChoiceD., They are carefully lined up with known sequences from other organisms.ChoiceB., The base sequences are aligned by matching short regions at the ends that overlap.

    How is the order of DNA fragments determined to obtain the sequence of the entire genome?

    Question 3 choices

    ChoiceA., They are sorted by size using gel electrophoresis.ChoiceB., The base sequences are aligned by matching short regions at the ends that overlap.ChoiceC., They are converted into sentences that are then assembled.ChoiceD., They are matched up to chromosomes prepared from the nucleus.ChoiceD., All of these choices are correct.

    How do repeated sequences in the genome complicate assembly of fragments?

    Question 4 choices

    ChoiceA., The repeated sequences may appear in many fragments.ChoiceB., Fragments from different regions of the chromosomes may appear identical if they contain the same repeated sequence.ChoiceC., Regions of sequence overlap may occur between fragments not actually adjacent in the genome.ChoiceD., All of these choices are correct.

    gene; DNA strand; chromosome; genome

    Select the answer option that lists the levels of genetic information in order from smallest to largest.

    gene; DNA strand; chromosome; genome

    gene; chromosome; DNA strand; genome

    DNA strand; gene; genome; chromosome

    DNA strand; chromosome; gene; genome

    genome; gene; chromosome; DNA strand

    True

    Sequencing reads can be aligned to a reference genome (i.e., human genome) to identify single nucleotide variants and potential mutations.

    True or False

    breaking the DNA into small fragments

    Which one of the following steps comes FIRST in shotgun sequencing?

    matching regions of overlap

    sequencing the DNA

    breaking the DNA into small fragments

    reconstructing the long sequence of nucleotides

    putting the sequences in the correct order

    True

    In DNA sequencing, the newly synthesized DNA strand that is complementary and anti-parallel to a template DNA strand is called a sequence read.

    True or False False

    To maintain the accuracy of the genome sequence, the DNA should not be cut into small pieces before sequencing.

    True or False

    sequences containing repeats longer than the DNA fragments to assemble

    Repeated DNA sequences represent a special challenge in genome sequence assembly. Which of the following would be harder to assemble correctly, assuming the number of copies of the repeat can be determined?

    sequences containing repeats shorter than the DNA fragments to assemble

    sequences containing repeats longer than the DNA fragments to assemble

    identical twins

    Which one of the following pairs of people has the exact same genome?

    identical twins fraternal twins mother and daughter father and son

    None of the other answer options is correct.

    False

    All DNA sequences are transcribed into RNA.

    True or False

    More sequences are conserved between rabbits and humans than between humans and mice.

    Source : quizlet.com

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