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MCAT Biochemistry > Ch 6 - DNA and Biotech > Flashcards

Ch 6 - DNA and Biotech Flashcards

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MCAT flashcards
1
Q

Nucleosides and Nucleotides

A
  • 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
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2
Q

Sugar phosphate backbone

A
  • 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
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3
Q

Purines and Pyrimidines

A
  • 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
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4
Q

Watson-Crick Model

A
  • 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
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5
Q

Chargaffs rule

A
  • used to determine percent of nucleotides in DNA strand
  • all 4 must add to 100%
  • A% = T%
  • C% = G%
  • total purines = total pyrimidines
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6
Q

Denaturation and Reannealing

A
  • denature by disrupting hydrogen bonds and making single strands
  • heat, basic pH, formaldehyde, urea
  • Reannealed - bring single strands together
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7
Q

Histones

A
  • 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
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8
Q

Nucleoprotein

A
  • example - histones
  • proteins associated with DNA
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9
Q

Heterochromatin and Euchromatin

A
  • heterochromatin - compacted chromatin
    • dark under light microscopy
    • not transcribed
  • Euchromatin - dispersed chromatin
    • genetically active DNA
    • light under microscopy
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10
Q

Telomere and Centromere

A
  • 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
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11
Q

DNA strand seperation

A
  • replisome helps DNA polymerase
  • Bidirectional unwinding
  • Origins of replication
    • multiple on one strand
    • single origin in bacteria
  • Replication fork on both sides of origin
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12
Q

Helicase

A
  • 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
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13
Q

Synthesis in DNA replication

A
  • 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
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14
Q

DNA polymerase in Eukaryote

A
  • 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
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15
Q

Cancer (DNA repair)

A
  • 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
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16
Q

Proofreading

A
  • 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
17
Q

Nucleotide and Base excision repair

A
  • 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
18
Q

Recombinant DNA

A
  • aka DNA cloning or polymerase chain reaction (PCR)
19
Q

DNA cloning

A
  • 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
20
Q

Restriction enzymes

A
  • 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
21
Q

DNA libraries

A
  • 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
22
Q

Hybridization

A
  • joining of complementary base pair sequences
  • DNA-DNA or DNA-RNA
  • uses 2 single stranded sequences
23
Q

PCR

A
  • 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
24
Q

Gel Electrophoresis

A
  • separate macromolecules via size and charge
  • all DNA is negative
  • migrates toward anode
  • longer sequence takes longer to migrate
25
Q

Southern Blot

A
  • 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
26
Q

DNA sequencing

A
  • 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
27
Q

Gene therapy

A
  • 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
28
Q

Transgenic and Knockout mice

A
  • 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

MCAT Biochemistry (10 decks)

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