Ch 6 - DNA and Biotech

Question 1

Nucleosides and Nucleotides

Answer 1

• nucelosides - 5 carbon sugars bound to nitrogenous bases via the C1 on sugar
• nucleotides - one or more phosphate groups attached to c5 of a nucleoside
  
  • named by how many phosphates
  • ex. adenosine triphosphate
• nucleic acids - strings of nucleotides
  
  • RNA - ribose sugar backbone
  • DNA - deoxyribose sugar backbone
    
    • 2 -OH groups replaced with H
• bases - adenine, guanine, cytosine, uracil, thymine
• nucleosides (ribose) - adenosine, guanosine, cytidine, uridine
• with deoxyribose - deoxyadenosine, deoxyguanosine, deoxyuridine, deoxythymidine

Question 2

Sugar phosphate backbone

Answer 2

• alternate sugar and phosphate
• always read 5’ to 3’
• 5’ has phosphate group attached to C5 on the sugar
• 3’ attaches phosphate to C3 on sugar
• Overall negative charge due to phosphate

Question 3

Purines and Pyrimidines

Answer 3

• purines - 2 rings
  
  • adenine and guanine
• pyrimidines - one ring
  
  • cytosine, thrymine, uracil
• aromatic
  
  • cyclic and planar
  • very stable and unreactive
  • delocalized electrons that from 2 ring like clouds
  • more stable due to nitrogen in ring

Question 4

Watson-Crick Model

Answer 4

• double helix in DNA
• antiparallel with respect to 5’ and 3’
• sugar phosphate backbone outside of helix, bases on inside
• complementary base pairing
  
  • A-T via 2 hydrogen bonds
  • C-G via 3 hydrogen bonds (stronger)
• B-DNA is right handed helix
  
  • turn every 3.4nm containing 10 base pairs
• Z-DNA is zigzag and left handed helix
  
  • turn every 4.6nm and 12 base pairs
  • high GC content
  • unstable, no known biological activity

Question 5

Chargaffs rule

Answer 5

• used to determine percent of nucleotides in DNA strand
• all 4 must add to 100%
• A% = T%
• C% = G%
• total purines = total pyrimidines

Question 6

Denaturation and Reannealing

Answer 6

• denature by disrupting hydrogen bonds and making single strands
• heat, basic pH, formaldehyde, urea
• Reannealed - bring single strands together

Question 7

Histones

Answer 7

• chromatin - series of nucleosomes, compact DNA wound around proteins
• nucleosomes - DNA wrapped around histones
• histones - proteins that are used to form nucleosomes
  
  • H2A, H2B, H3, H4 2 copies each to make a histone core
  • 200 bases around the core to form a nucleosome
  • H1, histone that seals off DNA. Adds stability

Question 8

Nucleoprotein

Answer 8

• example - histones
• proteins associated with DNA

Question 9

Heterochromatin and Euchromatin

Answer 9

• heterochromatin - compacted chromatin
  
  • dark under light microscopy
  • not transcribed
• Euchromatin - dispersed chromatin
  
  • genetically active DNA
  • light under microscopy

Question 10

Telomere and Centromere

Answer 10

• Telomere - repeating unit at end of DNA
  
  • Telomerase - enzyme that replaces last ends on telomeres
  • Progressive shortening of telomeres contributes to aging
  • high GC content prevents unraveling
• Centromeres - center of chromosome
  
  • heterochromatin and tandem repeat sequences with high GC content
  • connect sister chromatids

Question 11

DNA strand seperation

Answer 11

• replisome helps DNA polymerase
• Bidirectional unwinding
• Origins of replication
  
  • multiple on one strand
  • single origin in bacteria
• Replication fork on both sides of origin

Question 12

Helicase

Answer 12

• helicase - unwinds DNA ahead of polymerase
• single stranded DNA binding proteins bind to unraveled strand and prevent reassociation and degredation
• nuclease - degrades
• supercoiling - caused by unwinding of a helix
  
  • DNA topoisomerase - alleviates supercoiling by nicking one or both strands to allow relaxation. Then reseals
    
    • aka DNA gyrase
• Parental strands - template for daughter strands
  
  • semiconservative

Question 13

Synthesis in DNA replication

Answer 13

• DNA polymerase reads 3’-5’ and synthesizes complementary in 5’-3’ direction
• leading strand - copied continuously
• lagging strand - copied in opposite direction
  
  • okazaki fragments produced
• step 1: RNA primer via primase, several on the lagging strand
• step 2: DNA polymerase III (prokaryotes) and DNA polymerase (eukaryotes - alpha, delta, epsilon)
  
  • synthesize in 5’-3’ using 5’ deoxyribonucleotide triphosphates (dATP, dCTP, dTTP, dGTTP)
  • free pryophosphate released
• step 3: DNA polymerase I (pro) or RNase H (euk) removes RNA primer
• step 4: DNA polymerase I (pro) or DNA polymerase delta (euk) adds DNA where primer was
• step 5: DNA ligase seals ends of DNA segments together

Question 14

DNA polymerase in Eukaryote

Answer 14

• alpha, delta, episilon - synthesize leading and lagging strands
• delta - fill in DNA where RNA primer was
• Gamma - replicate mitochondrial DNA
• Beta and epsilon - DNA repair
• Delta and epsilon - help form sliding clamp. Strenghten interaction with template strand

Question 15

Cancer (DNA repair)

Answer 15

• cancer - cells proliferate excessively
  
  • accumulate mutations
• metastasis - migrate to distant tissues
• oncogenes - mutated genes that cause cancer
  
  • proto-oncogenes - before they are mutates
  • rapid cell cyle advancement
  • only one allele needs to be mutated
• Tumor suppressor genes (p53) - encode proteins that inhibit cell cycle or DNA repair processes
  
  • antioncogenes
  • promote cancer
  • both alleles must be mutated
    
    • multiple mutations required

Question 16

Proofreading

Answer 16

• proofreading function in the DNA polymerase enzyme
• lack of stability between hydrogen bonds is detected
• base excised and replaced
• use methylation to determine template strand
  
  • template strand more methylated due to being in the cell longer
• DNA ligase does not proofread - lagging strand more likely to have mutation
• Mismatch repair - Occurs in G₂ phase of cell cycle. Detect and remove errors due to replication
  
  • added check after proofreading

Question 17

Nucleotide and Base excision repair

Answer 17

• noticed in G1 and G2 of cell cycle
• nucleotide excision remair - UV light caused dimers of thymine
  
  • distorts shape of DNA
  • nucleotide excision repair
  • bulge in DNA noticed
  • excision endonuclease nicks backbone and removes dimer
  • DNA polymerase fills gap
  • DNA ligase fills gap
• base excision repair - cytosine deamination
  
  • loss amino group on cytosine - becomes uracil
  • uracil easily detected in DNA
  • removed by glycosylase enzyme
    
    • makes apurinic/apyrimidinic (AP) site or abasic site
  • AP endonuclease removes damaged DNA
  • DNA polymerase and DNA ligase fix

Question 18

Recombinant DNA

Answer 18

• aka DNA cloning or polymerase chain reaction (PCR)

Question 19

DNA cloning

Answer 19

• produce large amounts of desired sequence
• vector - piece of nucleic acid of interest
  
  • makes recombinant vector, typically bacterial or viral plasmid
• Use bacteria or virus to grow more DNA or protein of interest
• lyse organism to obtain replicated recombinant vector
  
  • then release cloned DNA from the vector

Question 20

Restriction enzymes

Answer 20

• aka restriction endonucleases
• recognize specific dsDNA sequences
  
  • palindromic, so both strands are identical when aligned 5’-3’
  • some cut both strands, other leave sticky ends
  • sticky ends used to attach fragments to vector

Question 21

DNA libraries

Answer 21

• collection of known sequences
• genomic library - large fragments, include coding (exon) and noncoding (intron)
• cDNA - complementary - constructed via reverse transcribing processed mRNA
  
  • lacks noncoding regions
  • aka expression libraries
  • cDNA used for producing recombinant proteins and transgenic animals

Question 22

Hybridization

Answer 22

• joining of complementary base pair sequences
• DNA-DNA or DNA-RNA
• uses 2 single stranded sequences

Question 23

PCR

Answer 23

• polymerase chain reaction
• produce copies of DNA sequence, no bacteria
• DNA denatured, replicated, cooled (reannealing)
• Double amount each cycle
• Uses primers thare complementary to the DNA next to region of interest
• DNA polymerase from hot spring bacteria in order to survive the heat required for denature

Question 24

Gel Electrophoresis

Answer 24

• separate macromolecules via size and charge
• all DNA is negative
• migrates toward anode
• longer sequence takes longer to migrate

Question 25

Southern Blot

Answer 25

• detects presence and quantity of DNA strands
• restriction enzymes cut DNA, seperated by gel electrophoresis
• Transfer fragments while maintaining seperation
• probe - bind complementary sequence to form dsDNA
  
  • radioisotopes in probe

Question 26

DNA sequencing

Answer 26

• uses dideoxyribonucleotides (ddTTP, ddCTP, ddATP, ddGTP)
  
  • contain hydrogen at C-3 rather than hydroxyl
  • must be the end of the chain because polymerase cannot add another base
  • many fragments and each terminates at the modified base
  • fragments seperated by size via electrophoresis
  • last base of each fragment read

Question 27

Gene therapy

Answer 27

• treat inherited diseases
• transfer normal gene into affected tissues
• need efficient gene delivery vector
  
  • transfer cloned gene into target DNA
  • use modified virus that cannot replicate
  • risk of activating an oncogene
  • may also use restrovirus

Question 28

Transgenic and Knockout mice

Answer 28

• Transgenic mice - altered at germ line
  
  • introduce cloned gene into fertilized ova or embryonic stem cell
  • transgene - gene that is introduced
• knockout mice - gene intentionally deleted
• more useful for dominant gene effects
• chimera - have cells with transgene lineage AND cells with original genes
  
  • created by injecting into embryonic stem cells