Cell Biology Lecture 2.docx

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Biological Sciences
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Dan Riggs

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Cell Biology Lecture 2 Paraniya Balakumar Deoxyribonucleic acid (DNA) is a molecule encoding the genetic instructions used in the development and functioning of all known living organisms and many viruses  DNA is made of a double stranded, antiparallel helix  Complementary base pairing occurs by hydrogen bonding in which A-T and C-G bond with one another Early work on DNA which uses the physiochemical approaches include: 1. DNA is a polynucleotide chain 2. Estimates made of genome size 3. Values were “tiny yet enormous”  Humans: 3.5 picograms of DNA/haploid  Genome: approxiamately 3.2 billion base pairs 4. Base pairing rules established by analyzing base composition 5. DNA absorbs light in ultraviolet range 6. Renaturation experiments defined complexity Chargaff: (1950s) base composition differed between organisms  [A] = [T]  [G] = [C]  [A] + [T] doesn’t = [G] + [C] o This is because AT bonds are held by 2 hydrogen bonds o CG bonds are held by 3 hydrogen bonds Absorbance measured by a spectrophotometer  It works by recording the ring structures of DNA which absorb in the ultraviolet range  DNA absorptions maximum range is approximately 260 nm  Absorbance can be used to determine DNA concentration  Absorbance increases about 1.5X if DNA is denatured. Thus changes in absorbance reveal difference in % double stranded (ds) vs % single stranded (ss) o This is because of the “satellite dish” analogy o Single stranded DNA (ssDNA) is flexible and contains nitrogenous bases which can freely rotate about glycosidic bond o Double stranded DNA (dsDNA) is more rigid, nitrogenous bases are limited in the number of positions they can occupy Complexity is a measure of the number of unique (vs. repetitive) sequences that exist in a genome.  There are lots of repetitive DNA in genomes  DNA reannealig (and later use of hybridization techniques) is useful for determining aspects of complexity o Reannealing the process in which you get two strands of DNA together and form complimentary gene pairs 5’ A-T-T-C-A-T-G-C-A-T-T-A-G-G-C-T-A-T 3’ 3’ T-A-A-G-T-A-C-G-T-A-A-T-C-C-G-A-T-A 5’ Renaturation (reannealing) of denatured DNA depends on many factors, like ionic strength, length of fragments, temperature, time  These monitor the ability of strands to get back together again  There is a certain procedure used which consists of: i. Purify DNA, shear to average size of 1000-2000 base pairs ii. Denature (heat), then allow to reanneal at lower temperature = which allows it to reanneal iii. Measure the % reassociated (by absorption) over time iv. The length of time required for half of the molecules to reanneal is referred to as the Cot½, and is a measure of complexity o Cot is the concentration of the fragments and the time required to reanneal; Co = concentration, t= time  Similar patterns indicative of unique (non-repetitive) sequences  Cot value reflects complexity (genome size and number of each “type” of sequence)  MS-2, T4, and E.coli all have very similar patterns; unique genomes but its taken a bit more time for E.coli to reanneal because of the genome size o Genome size of E.coli is 4.3 million base pairs when compared to the others o MS-2 takes shorter time to renneal since there are more copies In complex eukaryotic genomes there are three discrete fractions: highly repeated, moderately repeated, and nonrepeated fraction A decent analogy for understanding Cot curves is by looking at matching socks: you have one black sock and need to find a pair: 1. IF drawer contains 500 socks and 400 are black, you’ll have no problem finding a pair quickly 2. IF draw contains 500 socks and 100 are black, takes somewhat longer to find a pair 3. IF drawer contains 500 socks and are black, takes much longer time to find a pair
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