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Selected Articles on Mutagenesis

MW Unger , SY Liu , and DE Rancourt

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination
Nucl. Acids Res. 27: 1480-1484.

Paul Gaytán , Jorge Yáñez , Filiberto Sánchez , and Xavier Soberón

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols
Nucl. Acids Res. 29: e9.

RD Kirsch , and E Joly

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes
Nucl. Acids Res. 26: 1848-1850.

Qing Lin , Sarah L. Donahue , Tracy Moore-Jarrett , Shang Cao , Anna B. Osipovich , and H. Earl Ruley

Mutagenesis of diploid mammalian genes by gene entrapment
Nucleic Acids Research Advance Access published on November 6, 2006, DOI 10.1093/nar/gkl728.
Nucl. Acids Res. 34: e139.

Igor Shevelev , Giuseppina Blanca , Giuseppe Villani , Kristijan Ramadan , Silvio Spadari , Ulrich Hübscher , and Giovanni Maga

Mutagenesis of human DNA polymerase : essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities
Nucl. Acids Res. 31: 6916-6925.

Jaesung Lee , and David L. Herrin

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth
Nucl. Acids Res. 31: 4361-4372.

Michael O’Connor , Wyan-Ching Mimi Lee , Anuj Mankad , Catherine L. Squires , and Albert E. Dahlberg

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable
Nucl. Acids Res. 29: 710-715.

Eun Ju Lee , Joel Glasgow , Sew-Fen Leu , Ali O. Belduz , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 the cyclic nucleotide binding pocket
Nucl. Acids Res. 22: 2894-2901.

M.J. Pillaire , A. Margot , G. Villani , A. Sarasin , M. Defias , and A. Gentil

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(ll) adduct placed on codon 13 of the human H-ras proto-oncogen
Nucl. Acids Res. 22: 2519-2524.

Pamela Anderson , Joseph Monforte , Richard Tritz , Steven Nesbitt , John Hearst , and Arnold Hamper

Mutagenesis of the hairpin ribozyme
Nucl. Acids Res. 22: 1096-1100.

Huey-Nan Wu , Jun-YU Lee , Huey-Wen Huang , Yi-shuian Huang , and Tung-Guang Hsueh

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme
Nucl. Acids Res. 21: 4193-4199.

Ali O. Belduz , Eun Ju Lee , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli targeting positions 72 and 82 of the cyclic nucleotide binding pocket
Nucl. Acids Res. 21: 1827-1835.

Huey-Nan Wu , and Zhi-Shun Huang

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA
Nucl. Acids Res. 20: 5937-5941.

V. Pletsa , A. Gentil , A. Margot , J. Armier , S.A. Kyrtopoulos , and A. Sarasin

Mutagenesis by 06 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells
Nucl. Acids Res. 20: 4897-4901.

Akira Sekiguchi , Yasuo Komatsu , Makoto Koizumi , and Eiko Ohtsuka

Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco rinspot virus and its binding to polyamines
Nucl. Acids Res. 19: 6833-6838.

Aidan J. Doherty , Andrew F. Worall , and Bernard A. Connolly

Mutagenesis of the DNA binding residues in bovine pancreatic DNase1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease
Nucl. Acids Res. 19: 6129-6132.

Christian Baron , Johann Heider , and August Böck

Mutagenesis of selC, the gene for the selenocysteine-insertlng tRNA-species in E.coli: effects on in vivo function
Nucl. Acids Res. 18: 6761-6766.

Candice C. Sheldon , and Robert H. Symons

Mutagenesis analysis of a self-cleaving RNA
Nucl. Acids Res. 17: 5679-5685.

Jacques Piette , Howard B. Gamper , Albert Van de Vorst , and John E. Hearst

Mutagenesis induced by site specifically placed 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts
Nucl. Acids Res. 16: 9961-9977.

G. Chua , L. Taricani , W. Stangle , and P. G. Young

Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe
Nucl. Acids Res. 28: e53.

MJ Singer , MA Podyminogin , MA Metcalf , MW Reed , DA Brown , HB Gamper , RB Meyer , and RM Wydro

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides
Nucl. Acids Res. 27: e38.

A Melnikov , and PJ Youngman

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
Nucl. Acids Res. 27: 1056-1062.

RM Tucker , and DT Burke

Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol
Nucl. Acids Res. 24: 3467-3468.

David P. Siderovski , Toshifumi Matsuyama , Elena Frigerio , Stephen Chui , Xia Min , Heather Erfle , Martin Sumner-Smith , Richard W. Bamett , and Tak W. Mak

Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat)
Nucl. Acids Res. 20: 5311-5320.

Xiubei Liao , David Selinger , Steven Althoff , Anne Chiang , Diana Hamilton , Min Ma , and Jo Ann Wise

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites
Nucl. Acids Res. 20: 1607-1615.

Yuhong Zhou , Xiaoping Zhang , and Richard H. Ebright

Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase
Nucl. Acids Res. 19: 6052.

Bruno A. Gaëta , Stephen J. Sharp , and Thomas S. Stewart

Saturation mutagenesis of the Drosophila tRNAArg gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation
Nucl. Acids Res. 18: 1541-1548.

Laurent Dulau , Alain Cheyrou , and Michel Aigle

Directed mutagenesis using PCR
Nucl. Acids Res. 17: 2873.

John M. Hinz , Robert S. Tebbs , Paul F. Wilson , Peter B. Nham , Edmund P. Salazar , Hatsumi Nagasawa , Salustra S. Urbin , Joel S. Bedford , and Larry H. Thompson

Repression of mutagenesis by Rad51D-mediated homologous recombination
Nucl. Acids Res. 34: 1358-1368.

Tuck Seng Wong , Kang Lan Tee , Berhard Hauer , and Ulrich Schwaneberg

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution
Nucl. Acids Res. 32: e26.

Gordana Maravic , Janusz M. Bujnicki , Marcin Feder , Sándor Pongor , and Mirna Flögel

Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein–RNA interactions
Nucl. Acids Res. 31: 4941-4949.

Fumi Nagatsugi , Shigeki Sasaki , Paul S. Miller , and Michael M. Seidman

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog
Nucl. Acids Res. 31: e31.

Assen Marintchev , Michael R. Gryk , and Gregory P. Mullen

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase ß
Nucl. Acids Res. 31: 580-588.

P. Soultanas , and D. B. Wigley

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus
Nucl. Acids Res. 30: 4051-4060.

MS Dillingham , P Soultanas , and DB Wigley

Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation
Nucl. Acids Res. 27: 3310-3317.

P Neuner , R Cortese , and P Monaci

Codon-based mutagenesis using dimer-phosphoramidites
Nucl. Acids Res. 26: 1223-1227.

B Hallet , DJ Sherratt , and F Hayes

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein
Nucl. Acids Res. 25: 1866-1867.

W Wu , C Palaniappan , RA Bambara , and PJ Fay

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro
Nucl. Acids Res. 24: 1710-1718.

Gil Barzilay , Lisa J. Walker , Craig N. Robson , and Ian D. Hickson

Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity
Nucl. Acids Res. 23: 1544-1550.

Chung-Nam Chung , Yasushi Hamaguchi , Tasuku Honjo , and Masashi Kawaichi

Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jx
Nucl. Acids Res. 22: 2938-2944.

May 9, 2008

DNA Forensics -- Read Interesting Cases

Some Interesting Uses of DNA Forensic Identification

Identifying September 11th Victims Identifying the victims of the September 11, 2001, World Trade Center attack presented a unique forensic challenge because the number and identity of the victims were unknown and many victims were represented only by bone and tissue fragments. At the time of the attack, no systems were in place for rapidly identifying victims in disasters with more than 500 fatalities. The National Institutes of Justice assembled a panel of experts from the National Institutes of Health and other institutions to develop processes to identify victims using DNA collected at the site. Panel members produced forms and kits needed to enable the medical examiner’s office to collect reference DNA from victims’ previously stored medical specimens. These specimens were collected and entered into a database. The medical examiner's office also received about 20,000 pieces of human remains from the World Trade Center site, and a database of the victims’ DNA profiles was created. New information technology infrastructure was developed for data transfer between the state police and medical examiner’s office and to interconnect the databases and analytical tools used by panel members. In 2005 the search was declared at an end because many of the unidentified remains were too small or too damaged to be identified by the DNA extraction methods available at that time. Remains of only 1585, of the 2792 people known to have died had been identified. In 2007, the medical examiner's office reopened the search after the Bode Technology Group developed a new methodology of DNA extraction that required much less sample material than previously necessary. The victim DNA database and the new methods have allowed more victims to be identified, and further identifications will be possible as forensic DNA technology improves.

The DNA Shoah ProjectThe DNA Shoah Project is a genetic database of people who lost family during the Holocaust. The database will serve to reunite families separated during wartime and aid in identifying victims who remain buried anonymously throughout Europe.

Disappeared Children in ArgentinaNumerous people (known as "the Disappeared") were kidnapped and murdered in Argentina in the 1970s. Many were pregnant. Their children were taken at birth and, along with other kidnapped children, were raised by their kidnappers. The grandparents of these children have been looking for them for many years. Read an article about a DNA researcher who has been helping them.

Tomb of the Unknowns
Son of Louis XVI and Marie AntionettePARIS, Apr 19, 2000 (Reuters) -- Scientists cracked one of the great mysteries of European history by using DNA tests to prove that the son of executed French King Louis XVI and Marie-Antoinette died in prison as a child. Royalists have argued for 205 years over whether Louis-Charles de France perished in 1795 in a grim Paris prison or managed to escape the clutches of the French Revolution. In December 1999, the presumed heart of the child king was removed from its resting place to enable scientists to compare its DNA makeup with samples from living and dead members of the royal family -- including a lock of his mother Marie-Antoinette's hair.
The Murdered Nicholas Romanov, the Last Czar of Russia, and His Family

Peruvian Ice MaidenThe Ice Maiden was a 12-to-14-year old girl sacrificed by Inca priests 500 years ago to satisfy the mountain gods of the Inca people. She was discovered in 1995 by climbers on Mt. Ampato in the Peruvian Andes. She is perhaps the best preserved mummy found in the Andes because she was in a frozen state. Analysis of the Ice Maiden's DNA offers a wonderful opportunity for understanding her genetic origin. If we could extract mitochondrial DNA from the Ice Maiden's tissue and successfully amplify and sequence it, then we could begin to trace her maternal line of descent and possibly locate past and current relatives.

African Lemba Tribesmen In southern Africa, a people known as the Lemba heed the call of the shofar. They have believed for generations that they are Jews, direct descendants of the biblical patriarchs Abraham, Isaac, and Jacob. However unlikely the Lemba's claims may seem, modern science is finding ways to test them. The ever-growing understanding of human genetics is revealing connections between peoples that have never been seen before.

Super Bowl XXXIV Footballs and 2000 Summer Olympic SouvenirsThe NFL used DNA technology to tag all the Super Bowl XXXIV balls, ensuring their authenticity for years to come and helping to combat the growing epidemic of sports memorabilia fraud. The footballs were marked with an invisible, yet permanent, strand of synthetic DNA. The DNA strand is unique and is verifiable any time in the future using a specially calibrated laser.

A section of human genetic code taken from several unnamed Australian athletes was added to ink used to mark all official goods — everything from caps to socks — from the 2000 Summer Olympic Games. The technology is used as a way to mark artwork or one-of-a-kind sports souvenirs.

Migration PatternsEvolutionarily stable mitochondrial DNA and Y chromosomes have allowed bioanthropologists to begin to trace human migration patterns around the world and identify family lineage
See Genetic Anthropology, Ancestry, and Ancient Human Migrations
Wine Heritage Using DNA fingerprinting techniques akin to those used to solve crimes and settle paternity suits, scientists at the University of California, Davis, have discovered that 18 of the world's most renowned grapevine varieties, or cultivars are close relatives. These include varieties long grown in northeastern France such as Chardonnay, the "king of whites," and reds such as Pinot and Gamay noir, are close relatives.

DNA Banks for Endangered Animal Species

Poached Animals

Declining Grizzly Bear Population
Snowball the Cat A woman was murdered in Prince Edward Island, Canada. Her estranged husband was implicated because a snowy white cat hair was found in a jacket near the scene of the crime, and DNA fragments from the hair matched DNA fragments from Snowball, the cat belonging to the husband's parents. See M. Menotti-Raymond et al., "Pet cat hair implicates murder suspect," Nature, 386, 774, 1997. Also see Holmes, Judy, Feline Forensics, Syracuse University Magazine, Summer 2001.

Angiosperm Witness for the Prosecution The first case in which a murderer was convicted on plant DNA evidence was described in the PBS TV series, "Scientific American Frontiers." A young woman was murdered in Phoenix, Arizona, and a pager found at the scene of the crime led the police to a prime suspect. He admitted picking up the victim but claimed she had robbed him of his wallet and pager. The forensic squad examined the suspect's pickup truck and collected pods later identified as the fruits of the palo verde tree (Cercidium spp.). One detective went back to the murder scene and found several Palo Verde trees, one of which showed damage that could have been caused by a vehicle. The detective's superior officer innocently suggested the possibility of linking the fruits and the tree by using DNA comparison, not realizing that this had never been done before. Several researchers were contacted before a geneticist at the University of Arizona in Tucson agreed to take on the case. Of course, it was crucial to establish evidence that would stand up in court on whether individual plants (especially Palo Verde trees) have unique patterns of DNA. A preliminary study on samples from different trees at the murder scene and elsewhere quickly established that each Palo Verde tree is unique in its DNA pattern. It was then a simple matter to link the pods from the suspect's truck to the damaged tree at the murder scene and obtain a conviction. [WNED-TV (PBS - Buffalo, N.Y.)]

DNA Forensics - DNA Fingerpring

How does forensic identification work?

Any type of organism can be identified by examination of DNA sequences unique to that species. Identifying individuals within a species is less precise at this time, although when DNA sequencing technologies progress farther, direct comparison of very large DNA segments, and possibly even whole genomes, will become feasible and practical and will allow precise individual identification.

To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of regions.

Some Examples of DNA Uses for Forensic Identification

Identify potential suspects whose DNA may match evidence left at crime scenes

Exonerate persons wrongly accused of crimes
Identify crime and catastrophe victims
Establish paternity and other family relationships
Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers)

Detect bacteria and other organisms that may pollute air, water, soil, and food

Match organ donors with recipients in transplant programs
Determine pedigree for seed or livestock breeds
Authenticate consumables such as caviar and wine

Is DNA effective in identifying persons?

[answer provided by Daniel Drell of the U.S. DOE Human Genome Program]

DNA identification can be quite effective if used intelligently. Portions of the DNA sequence that vary the most among humans must be used; also, portions must be large enough to overcome the fact that human mating is not absolutely random.

Consider the scenario of a crime scene investigation . . .
Assume that type O blood is found at the crime scene. Type O occurs in about 45% of Americans. If investigators type only for ABO, finding that the "suspect" in a crime is type O really doesn't reveal very much.
If, in addition to being type O, the suspect is a blond, and blond hair is found at the crime scene, you now have two bits of evidence to suggest who really did it. However, there are a lot of Type O blonds out there.
If you find that the crime scene has footprints from a pair of Nike Air Jordans (with a distinctive tread design) and the suspect, in addition to being type O and blond, is also wearing Air Jordans with the same tread design, you are much closer to linking the suspect with the crime scene.
In this way, by accumulating bits of linking evidence in a chain, where each bit by itself isn't very strong but the set of all of them together is very strong, you can argue that your suspect really is the right person.
With DNA, the same kind of thinking is used; you can look for matches (based on sequence or on numbers of small repeating units of DNA sequence) at many different locations on the person's genome; one or two (even three) aren't enough to be confident that the suspect is the right one, but four (sometimes five) are used. A match at all five is rare enough that you (or a prosecutor or a jury) can be very confident ("beyond a reasonable doubt") that the right person is accused.

How is DNA typing done?

Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products.

In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions (markers).

Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. A marker by itself usually is not unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person.
If the sample profiles don't match, the person did not contribute the DNA at the crime scene.

If the patterns match, the suspect may have contributed the evidence sample. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim. The question is, How small do the odds have to be when conviction of the guilty or acquittal of the innocent lies in the balance? Many judges consider this a matter for a jury to take into consideration along with other evidence in the case. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are about 50:50.

The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. Four to six probes are recommended. Testing with several more probes will become routine, observed John Hicks (Alabama State Department of Forensic Services). He predicted that DNA chip technology (in which thousands of short DNA sequences are embedded in a tiny chip) will enable much more rapid, inexpensive analyses using many more probes and raising the odds against coincidental matches.

What are some of the DNA technologies used in forensic investigations?

Restriction Fragment Length Polymorphism (RFLP)

RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample.

RFLP was one of the first applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA-analysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with RFLP.

PCR Analysis

Polymerase chain reaction (PCR) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.

STR Analysis

Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in a billion.

Mitochondrial DNA Analysis

Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable.

All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing-person investigations.

Y-Chromosome Analysis

The Y chromosome is passed directly from father to son, so analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors.

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Jun 12, 2008

CGH Protocols

Production Sequencing Protocols

Roe Lab. Molecular Biology Protocols

May 21, 2008

DNA (Gene) Transformation Protocols

Selected Articles on Mutagenesis

May 9, 2008

DNA Forensics -- Read Interesting Cases

DNA Forensics - DNA Fingerpring

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DNA from the Beginning

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