DNA. Chapter 1. Molecular Diagnostics Fundamentals, Methods and Clinical Applications Second Edition 1/29/2013. Copyright 2012 F.A.

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Kristopher Cooper
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1 DNA Chapter 1 1

There are four common nitrogen bases; two with purine ring structures (adenine and guanine) and two with pyrimidine

2 Deoxyribonucleic acid (DNA) is a genetic information storage system. A T G C T A C G DNA is a polymer of nucleotides. Nucleotides are phosphorylated nucleosides. DNA Nucleosides comprise a ribose sugar and a nitrogen base. There are four common nitrogen bases; two with purine ring structures (adenine and guanine) and two with pyrimidine ring structures (thymine and cytosine). The order or sequence of nucleotides in the DNA polymer is the code for information storage. 2

3 Formation of nucleotide by removal of H 2 O Glycosidic bond between 2'- deoxyribose and base Phosphormonoester bond between phosphate and 2'- deoxyribose Deoxyribonucleotides Deoxyguanosine Deoxyadenosine Deoxythymidine Deoxycytidine phosphate phosphate phosphate phosphate dg da dt dc Nucleic acid sequence.dgactcctgctagctacgtagccta... 3

4 Polynucleotide chain Nucleotides are joined to each other in polynucleotide chains 3' 5' phosphodiester linkage 4

5 DNA exists as two polymers joined together by hydrogen bonds. cytosine Two hydrogen bonds form between A and T. Three hydrogen bonds form between C and G. 5

6 The two polymers form a double helix. 6

7 Polarity DNA strands have polarity based on the phosphodiester backbone Nucleotides are joined through the ribose sugars and phosphate groups. From previous base To next base DNA strands have 5 to 3 polarity. 7

8 DNA is usually a right handed double helix The double helix exists in multiple conformations B DNA Physiological condition 10 bp per turn A DNA Low humidity 11 bp per turn Z DNA High conc. of positive charged ions 12 bp per turn 8

Covalently closed, circular DNA (cccdna) Because there are no interruptions in either polynucleotide chain, the two strands of cccdna cannot be separated from each other without the breaking of

9 Covalently closed, circular DNA (cccdna) Because there are no interruptions in either polynucleotide chain, the two strands of cccdna cannot be separated from each other without the breaking of covalent bond. If we wish to separate the two circular strands without permanently breaking any bonds in the sugarphosphate backbones, we have to pass one strand through the other strand repeatedly. The number of times one strand would have to be passed through the other strand in order for the two strand to be entirely separated from each other is called the linking number. The linking number is an invariant topological property of cccdna. How can we remove supercoils from cccdna if it is not already relaxed? DNase I treatment break the phosphodiester bond. After nick, DNA can freely rotate. 9

The greater the writhe, the more compact of a cccdna.

10 Covalently closed, circular DNAs of the same length but of different linking number are called DNA topoisomers. The greater the writhe, the more compact of a cccdna. DNA topoisomers 1 relaxed (nicked circular) DNA 2 linear DNA 3 highly supercoiled cccdna Separation of relaxed and supercoiled DNA by gel electrophoresis 10

11 DNA Replication? Conservative Semiconservative Dispersive DNA Replication 5 3 Phosphodiester bond 3 hydroxyl group Pyrophosphate 5 phosphate group 5 3 A new DNA strand is polymerized in the 5 to 3 direction, reading the parent strand in the 3 to 5 direction. 11

hydroxyl group at the 3 end of the primer strand

12 The mechanism of DNA synthesis The phosphordiester bond is formed in an SN2 reaction in which the hydroxyl group at the 3 end of the primer strand attacks the phosphoryl group of the incoming nucleoside triphosphate. 12

13 DNA Replication OP5 - GTAGCTCGCTGAT - 3 OH OH3 - CATCGAGCGACTA - 5 OP The two strands of the resulting double helix are antiparallel. DNA Replication Despite the antiparallel nature of the two strands, DNA synthesis proceeds along both strands in the same direction. 13

14 The replication fork The junction between the newly separated template strands and the unreplicated duplex DNA is replication fork. The replisome is a group of proteins required for DNA replication. Lagging strand 3 5 Helicase Primase Leading strand Polymerase 14

Each Okazaki fragment requires one RNA primer. RNA primer It s a DNA repair event. RNAase H (hybrid) recognizes and removes most of RNA primer.

15 All DNA polymerases require a primer with a free 3 OH. Start new RNA chain de novo. Primase is a specialized RNA polymerase dedicated to making short RNA primers on an ssdna template. One RNA primer is required for the leading strand. Each Okazaki fragment requires one RNA primer. RNA primer It s a DNA repair event. RNAase H (hybrid) recognizes and removes most of RNA primer. RNAase H can only cleave bonds between two ribonucleotides. Exonuclease removes the final ribonucleotide that linked to the DNA end. DNA ligase use ATP to create a phosphordiester bond between the 3 OH and 5 phosphate of the repaired strand. Removal of RNA primer 15

16 Catalyze the separation of the two strands of duplex DNA at the replication fork. It binds to and moves directionally (polarity) along ssdna using the energy of ATP hydrolysis to displace any DNA strand that is annealed to the bound ssdna. DNA helicase act processively. It binds to lagging strand at the replication fork. DNA helicase Single stranded binding proteins Stabilize the separated strands. Cooperative binding binding of one SSB promotes the binding of another SSB to the immediately adjacent ssdna. SSB interacts with ssdna through electrostatic interactions with the phosphate backbone and stacking interactions with the DNA bases. 16

17 Two types of function are needed to convert double stranded DNA to the single stranded DNA Helicase Catalyze the separation of the two strands of duplex DNA Topo I : nick one strand of double helix topo II (gyrase) : introduce coiling by cutting both strands of helix, passing another part of duplex and re ligating the cut strand SSB Stabilize the separated strands. Action of topoisomerase at the replication fork As the strands of DNA are separated at the replication fork, the double stranded DNA in front of the fork becomes increasingly positively supercoiled. If DNA strands remain unbroken, linking number will not change. Smaller number of base pairs need to accommodate the same linking number. 17

DNA Metabolizing Enzymes Polymerases: catalyze formation of the phosphodiester bond Helicases: unwind and untangle Primase: synthesizes a short ribonucleic acid (RNA) to prime DNA synthesis

18 DNA Metabolizing Enzymes Polymerases: catalyze formation of the phosphodiester bond Helicases: unwind and untangle Primase: synthesizes a short ribonucleic acid (RNA) to prime DNA synthesis Methylases: add methyl groups to nitrogen bases Deaminases: take amino groups from nitrogen bases Nucleases: cut DNA Ligases: catalyze formation of a single phosphodiester bond DNA polymerase I 103 kd Proteolytic treatment Klenow fragment 68kD Polymerase 3 5 exonuclease activities 35 kd 5 3 exonuclease activities Nick translation 18

19 Nucleases DNA Metabolizing Enzymes Exonucleases: remove bases from the ends of DNA strands Endonucleases: cut DNA strands internally Restriction Endonucleases Enzyme Isolated From Recognition Sequence Eco RI E. coli, strain R, 1st enzyme Gv AATTC Eco RV E. coli, strain R, 5th enzyme Gv ATATC Hind III H. influenzae, strain d, 3rd enzyme Av AGCTT 19

20 Restriction endonucleases cut doublestranded DNA. 5 G AATTC CTTAA G 5 5 CCC GGG GGG CCC 5 CTGCA G G ACGTC 5 5 EcoR1 5 overhang Sma1 blunt PstI 3 overhang DNA Ligase PstI cut Ligated Recut DNA ligase pastes or ligates cut fragments of DNA together. 20

21 Plasmids Plasmids are used to move genes from cell to cell. Classes of Naturally Occurring Plasmids Large plasmids ( kb) F factor, R plasmids Conjugative = selftransmissible 1 2 per chromosome Small plasmids ( kb) Some R factors Nonconjugative per chromosome 21

22 Recombinant DNA Technology Required to perform recombinant DNA technology: Plasmid vectors: for carrying DNA Restriction enzymes: for cutting DNA DNA ligase: for pasting DNA Cloning of a Gene A plasmid, restriction enzymes, and DNA ligase are used to clone genes. 22

23 Recombination Gene cloning is a method of in vitro recombination. Recombination occurs in vivo by crossing over random assortment of chromosomes. Duplicated diploid chromosomes in parent Gametes will merge to form new diploid individual. Recombinants Recombinant organisms are naturally produced in eukaryotes through sexual reproduction. Recombinant organisms are naturally produced in prokaryotes by gene transfer through conjugation, transduction, or transformation. Gametes Zygote x Donor Conjugation Transduction Transformation Recipient 23

24 Recombinant bacteria can be detected by growth on selective media. + + met - bio - thr - leu No growth on medium without met, bio, thr, leu: minimal medium (auxotrophic) Grows on minimal medium (prototrophic) Conjugation F- F+ Hfr Conjugation Integration Loss Detachment F Abnormal detachment 24

25 Transduction Bacteriophage DNA Bacterial chromosome Phagecarrying bacterial gene Recombinant New host Lysed bacterium Transformation Plasmid DNA or fragmented from lysed bacterium Uptake and integration Recombinant 25

26 Summary DNA is a polymer of nucleotides, storing genetic information in the order of the nucleotide sequence. DNA replication conserves the DNA sequence. The two strands of double stranded DNA are antiparallel, and complementary DNA can be manipulated in vitro using DNAmetabolizing enzymes. Recombination is a natural process in eukaryotes and prokaryotes to produce offspring with new genetic combinations (recombinants). Restriction endonucleases, ligase, and plasmids are used to make new genetic combinations in vitro. 26

CH 4 - DNA. DNA = deoxyribonucleic acid. DNA is the hereditary substance that is found in the nucleus of cells

CH 4 - DNA. DNA = deoxyribonucleic acid. DNA is the hereditary substance that is found in the nucleus of cells CH 4 - DNA DNA is the hereditary substance that is found in the nucleus of cells DNA = deoxyribonucleic acid» its structure was determined in the 1950 s (not too long ago).» scientists were already investigating