14: DNA Structure and Function Flashcards
Historical Basis of Modern Understanding, DNA Structure and Sequencing, Basics of DNA Replication, DNA Replication in Prokaryotes, DNA Replication in Eukaryotes, DNA Repair
How large is the human genome?
Each human cell has 23 pairs of chromosomes, one from each parent, and a mitochondrial genome, inherited exclusively from the mother. The human haploid genome contains 3 billion base pairs and has between 20,000 and 25,000 functional genes.
What is transformation?
The process in which external DNA is taken up by a cell.
Who discovered DNA?
In the 1860s, Friedrich Miescher, a physician by profession, was the first person to isolate phosphate-rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.
When was bacterial transformation discovered?
The first experiment, reported in 1928 by Frederick Griffith, a British bacteriologist, suggesting that bacteria are capable of transferring genetic information through a process known as transformation. He used Streptococcus pneumoniae, injecting mice with combinations of the R (non-pathogenic) and S (pathogenic) strains. The strains are so named depending on whether they have a Rough or Smooth appearance, which is caused be the absence or presence of a capsule. They survived with live R strains and heat-killed S strains, but died with a mixture of live R strain and heat-killed S strain. Upon isolating the live bacteria from the dead mouse, only the S strain was recovered. Injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle. These experiments are now famously known as Griffith’s transformation experiments.
When was DNA identified as the transforming principle?
An experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation, in an era when it had been widely believed that it was proteins that served the function of carrying genetic information (with the very word “protein” itself coined to indicate a belief that its function was primary). They isolated the S strain from the dead mice in Griffith’s experiment and isolated the proteins and nucleic acids (namely RNA and DNA), and conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.
When was the first application of DNA analysis in forensics?
DNA evidence was used for the first time to solve an immigration case. The story started with a teenage boy returning to London from Ghana to be with his mother. Immigration authorities at the airport were suspicious of him, thinking that he was traveling on a forged passport. After much persuasion, he was allowed to go live with his mother, but the immigration authorities did not drop the case against him. All types of evidence, including photographs, were provided to the authorities, but deportation proceedings were started nevertheless. Around the same time, Dr. Alec Jeffreys of Leicester University in the United Kingdom had invented a technique known as DNA fingerprinting. The immigration authorities approached Dr. Jeffreys for help. He took DNA samples from the mother and three of her children, plus an unrelated mother, and compared the samples with the boy’s DNA. Because the biological father was not in the picture, DNA from the three children was compared with the boy’s DNA. He found a match in the boy’s DNA for both the mother and his three siblings. He concluded that the boy was indeed the mother’s son.
What do forensic scientists analyze?
Forensic scientists analyze many items, including documents, handwriting, firearms, and biological samples. They analyze the DNA content of hair, semen, saliva, and blood, and compare it with a database of DNA profiles of known criminals.
What does forensic DNA analysis include?
Analysis includes DNA isolation, sequencing, and sequence analysis; most forensic DNA analysis involves polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci and electrophoresis to determine the length of the PCR-amplified fragment. Only mitochondrial DNA is sequenced for forensics.
Where do forensic scientists work?
Forensic scientists are expected to appear at court hearings to present their findings. They are usually employed in crime labs of city and state government agencies. Geneticists experimenting with DNA techniques also work for scientific and research organizations, pharmaceutical industries, and college and university labs.
What education is required to be a forensic scientist?
Students wishing to pursue a career as a forensic scientist should have at least a bachelor’s degree in chemistry, biology, or physics, and preferably some experience working in a laboratory.
When was DNA identified as genetic material?
A series of experiments conducted in 1952 by Alfred Hershey and Martha Chase that helped to confirm that DNA is genetic material. They worked with bacteriophages, labeling the protein coat in one batch of phage with radioactive sulfur, 35S, and labeling their DNA in another batch with radioactive phosphorus, 32P. After infection, the phage-bacterial suspensions were put in blenders, causing the phage coat to be detached from the host cell. The suspensions were then spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the 35S-labeled tube, the supernatant contained the radioactivity. In the 32P-labeled tube, the pellet contained the radioactivity. This suggested that it was DNA and not protein that was injected into the bacterial cells, providing evidence that DNA was the genetic material.
What is a bacteriophage?
A virus that infects and replicates within bacteria and archaea. It infects the host cell by attaching to its surface, and injecting its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages.
How are viruses structured?
Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA.
What are Chargaff’s rules?
Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. This is also known as Chargaff’s rules.
What is electrophoresis?
A technique used to separate DNA fragments according to size.
What are nucleotides?
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
What is a phosphodiester bond?
The nucleotides combine with each other by covalent bonds known as phosphodiester bonds or linkages. The carbon atoms of the five-carbon sugar are numbered 1’, 2’, 3’, 4’, and 5’ (1’ is read as “one prime”). The phosphate residue is attached to the hydroxyl group of the 5’ carbon of one sugar of one nucleotide and the hydroxyl group of the 3’ carbon of the sugar of the next nucleotide, thereby forming a 5’-3’ phosphodiester bond.
What are purines?
The purines have a double ring structure with a six-membered ring fused to a five-membered ring.
What are pyrimidines?
Pyrimidines are smaller in size than purines; they have a single six-membered ring structure.
Who was involved in the discovery of the structure of DNA?
In the 1950s, Francis Crick and James Watson worked together to determine the structure of DNA at the University of Cambridge, England. Other scientists like Linus Pauling and Maurice Wilkins were also actively exploring this field. Pauling had discovered the secondary structure of proteins using X-ray crystallography. In Wilkins’ lab, researcher Rosalind Franklin was using X-ray diffraction methods to understand the structure of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of Franklin’s data because Crick had also studied X-ray diffraction. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine. Unfortunately, by then Franklin had died, and Nobel prizes are not awarded posthumously.
What is the DNA double helix?
In this model, base-pairing occurs between a purine and pyrimidine (A-T and G-C), which are called complementary base pairs. The base pairs are stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds, and cytosine and guanine form three. The two strands are anti-parallel, where the 3’ end of one strand faces the 5’ end of the other. The sugar and phosphate of the nucleotides form the backbone of the structure, and the nitrogenous bases are inside. Each base pair is separated from the other by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Ten base pairs are present per turn of the helix. The diameter of the DNA double helix is 2 nm, and is uniform throughout. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves.
How have DNA sequencing techniques improved over time?
Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process. Using radiolabeled nucleotides also compounded the problem through safety concerns. With currently available technology and automated machines, the process is cheap, safer, and can be completed in a matter of hours. Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today.
What is a dideoxynucleotide?
A chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are known as 2’,3’ because both the 2’ and 3’ positions on the ribose lack hydroxyl groups (hence they differ from deoxyribonucleotides by the lack of a 3’ hydroxyl group), and are abbreviated as ddNTPs. If a ddNTP is added to a growing DNA strand, the chain is not extended any further because the free 3’ OH group needed to add another nucleotide is not available.
How does Sanger sequencing work?
A method of DNA sequencing developed by Fred Sanger based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes. It uses dye-labeled ddNTPs to generate DNA fragments that terminate at different points. The DNA is separated by capillary electrophoresis on the basis of size, and from the order of fragments formed, the DNA sequence can be read. The DNA sequence readout is shown on an electropherogram that is generated by a laser scanner. The DNA sample to be sequenced is denatured (separated into two strands) by heating it to high temperatures. The DNA is divided into four tubes in which a primer, DNA polymerase, and all four nucleotides (A, T, G, and C) are added. In addition to each of the four tubes, limited quantities of one of the four ddNTPs are added to each tube respectively. The tubes are labeled as A, T, G, and C according to the ddNTP added. For detection purposes, each of the four ddNTPs carries a different fluorescent label. Chain elongation continues until a fluorescent dideoxy nucleotide is incorporated, after which no further elongation takes place. After the reaction is over, electrophoresis is performed. Even a difference in length of a single base can be detected. The sequence is read from a laser scanner. For his work on DNA sequencing, Sanger received a Nobel Prize in chemistry in 1980.
