Examples of DNA sequencing in the following topics:
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- DNA sequencing techniques are used to determine the order of nucleotides (A,T,C,G) in a DNA molecule.
- However, until the 1990s, the sequencing of DNA was a relatively expensive and long process.
- A Sanger sequencing reaction is just a modified in vitro DNA replication reaction.
- As such the following components are needed: template DNA (which will the be DNA whose sequence will be determined), DNA Polymerase to catalyze the replication reactions, a primer that basepairs prior to the portion of the DNA you want to sequence, dNTPs, and ddNTPs.
- From the order of fragments formed, the DNA sequence can be read.
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- Noncoding DNA are sequences of DNA that do not encode protein sequences but can be transcribed to produce important regulatory molecules.
- In genomics and related disciplines, noncoding DNA sequences are components of an organism's DNA that do not encode protein sequences.
- However, many types of noncoding DNA sequences do have important biological functions, including the transcriptional and translational regulation of protein-coding sequences, origins of DNA replication, centromeres, telomeres, scaffold attachment regions (SARs), genes for functional RNAs, and many others.
- Other noncoding sequences have likely, but as-yet undetermined, functions.
- More than 98% of the human genome does not encode protein sequences, including most sequences within introns and most intergenic DNA.
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- In the shotgun sequencing method, several copies of a DNA fragment are cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun).
- By matching overlapping sequences at the end of each fragment, the entire DNA sequence can be reformed.
- This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.
- Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing, which is a group of automated techniques used for rapid DNA sequencing.
- When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops.
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- Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences.
- Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA.
- Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides.
- Southern blotting is used to find a particular sequence in a sample of DNA.
- DNA fragments are separated on a gel, transferred to a nylon membrane, and incubated with a DNA probe complementary to the sequence of interest.
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- Genome sequences and expression can be analyzed using DNA microarrays, which can contribute to detection of disease and genetic disorders.
- DNA microarrays are methods used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and identify sequences .
- Although the study of medical applications of genome sequencing is interesting, this discipline tends to dwell on abnormal gene function.
- It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely.
- DNA microarrays can be used to analyze gene expression within the genome.
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- Both the packaging of DNA around histone proteins, as well as chemical modifications to the DNA or proteins, can alter gene expression.
- The tags do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins .
- These changes to DNA are inherited from parent to offspring, such that while the DNA sequence is not altered, the pattern of gene expression is passed to the next generation.
- The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent.
- Nucleosomes can slide along DNA.
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- If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG.
- During DNA replication, each strand is copied, resulting in a daughter DNA double helix containing one parental DNA strand and a newly synthesized strand.
- A mutation is a change in the sequence of the nitrogen bases.
- For example, in the sequence AATTGGCC, a mutation may cause the second T to change to a G.
- Most of the time when this happens the DNA is able to fix itself and return the original base to the sequence.
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- The ends of the linear chromosomes are known as telomeres: repetitive sequences that code for no particular gene.
- In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times.
- After each round of DNA replication, some telomeric sequences are lost at the 5' end of the newly synthesized strand on each daughter DNA, but because these are noncoding sequences, their loss does not adversely affect the cell.
- However, even these sequences are not unlimited.
- After sufficient rounds of replication, all the telomeric repeats are lost, and the DNA risks losing coding sequences with subsequent rounds.
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- DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.
- Watson and Crick's discovery that DNA was a two-stranded double helix provided a hint as to how DNA is replicated.
- DNA from cells grown exclusively in 15N produced a lower band than DNA from cells grown exclusively in 14N.
- So each "daughter" DNA actually consists of one "old" DNA strand and one newly-synthesized strand.
- When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell.
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- In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III.
- DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.
- There are specific nucleotide sequences called origins of replication where replication begins.
- In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences.
- DNA polymerase I replaces the RNA primer with DNA.