Module 2 - DNA Flashcards
(117 cards)
1
Q
Biomolecules
A
make up cell, essential to biological processes, organic molecules
2
Q
Macromolecules
A
carbohydrates, lipids, proteins, nucleic acids
3
Q
Carbohydrate structure and function
A
- long chain carbs made up of polysaccharides e.g. lactose, sucrose
- polysaccharides made of monosaccharides e.g. glucose, fructose linked by glycosidic bonds
- provide energy to body through glucose
4
Q
Carb composition of cell wall + storage of bacteria
A
- peptidoglycan = cell wall - complex polysaccharide
- glycogen = storage
5
Q
Carb composition of cells wall + storage of plants
A
- cellulose + pectins = cell wall
- starch = storage
6
Q
Carb composition of cells wall of insects + fungi
A
- chitin = exoskeleton + cell wall
7
Q
Lipids structure and function
A
- include waxes, oils, fats, steroids, phospholipids
- insoluble in water, hydrophobic
- non-polar carbon bonds
- provide energy, insulation, building blocks of hormones
- phospholipids = components of cell membrane
- waxes = coat surfaces of plants + animal skin to prevent water loss
- complex lipids = signaling molecules, photoreceptors, hormones, pigments
8
Q
Structure and function of proteins
A
- polymers of 20 different amino acids
- linear sequences held by peptide bonds
- structure affects function
- functions = structural, regulatory, contractile, storage, transport, protective
- enzymes = catalysts or hormones = chemical signals that control processes
- denaturation = loss of function as protein altered due to temperature, pH or chemicals
9
Q
Nucleic acids structure and function
A
- store heritable genetic information + carry instructions for function of cell
- fundamental to heritability
- can be DNA or RNA - ribose sugar in RNA, deoxyribose sugar in DNA
- genetic information translated from nucleic acid to protein - use RNA as intermediate
- code information for amino acid sequence of proteins
- consist of polynucleotide strands
10
Q
DNA structure and function
A
- nucleotides with deoxyribose sugar, phosphate group + nitrogenous base
- forms chromosomes
- phosphate group + 5C sugar joined by phosphodiester bond to form sugar-phosphate backbone
- double helix - two polynucleotide strands connected by H-bonds between bases –> A-T = 2 H-bonds, C-G = 3 H-bonds
- antiparallel strands
11
Q
Nitrogenous bases
A
Adenine, thymine, guanine and cytosine
12
Q
Purines (single ring)
A
A + G
13
Q
Pyrimidines (double ring)
A
C + T + U
14
Q
RNA structure and function
A
- similar to DNA except contain ribose sugar and uracil instead of thymine
- single polynucleotide strand
- use information from DNA to specify sequence of amino acids
- mRNA = messenger that translates to amino acids
15
Q
DNA replication definition
A
biological process of producing two identical replicas of DNA from one original DNA molecule
16
Q
Directionality of DNA replication
A
- DNA polymerase adds nucleotides to 3’ end of strand
- DNA polymerase synthesizes 5’ to 3’ direction
17
Q
DNA strand being copied in DNA replication
A
- template strand
- nucleotides on single strand used to reconstruct nucleotides on newly synthesized partner strand
18
Q
DNA replication process
A
- intrastrand bonds weak = easy to separate strands
- initiation
- elongation
- termination
19
Q
Primase
A
synthesizes RNA primer required for DNA polymerase to add nucleotides to the strand
20
Q
Helicase
A
unwinds DNA helix, creating replication fork
21
Q
Leading strand
A
continuous, same direction replication fork moves
22
Q
Lagging strand
A
discontinuous, Okazaki fragments that each require a separate primer + joined by DNA ligase
23
Q
Single strand binding proteins
A
coat strands to keep them apart, stabilize and relax DNA
24
Q
Topoisomerases
A
- unwinding of DNA causes torsional strain which leads to supercoiling
- topoisomerases prevent supercoiling
- DNA gyrase used in DNA replication to relieve torsional strain
25
Eukaryotic DNA replication
- more complex
- larger amount of DNA in multiple chromosomes
- linear structure and telomeres
- more enzymes required
26
DNA cloning definition
artificial replication of DNA by using right selection of enzymes present in nature
27
DNA cloning process
- cut DNA at sequence specific sites
- ligate DNA fragments together to form recombinant DNA
- amplify DNA as plasmids in bacteria
- separate and visualize DNA for research
28
Restriction endonucleases
- enzymes that cleave DNA at specific sites
- each enzyme has different restriction site, some produce sticky ends
- recombinant DNA = cutting DNA fragments from different organisms with same nuclease to produce complementary sticky ends joined together with ligase
29
Recombinant DNA replication
- stably replicated using host e.g. E. coli
- fragment of interest ligated into bacterial plasmid
- plasmid introduced into E. coli by e-shock or electrocorporation
- bacteria incorporate DNA into genome where it will replicate within normal replication process of cell
30
Gel electrophoresis
- allows separation of DNA fragments by size
- gel made of agarose
- submersed in buffe that carries current
- subjected to electrical field
- DNA migrates to positive pole
- large fragments move slower than small fragments
- DNA visualized with fluorescent dye
- fragments cut from gel + purified for use in recombinant DNA
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Mitosis definition
cell division that produces 2 genetically identical daughter cells
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Chromatin
mix of DNA and proteins, forms chromosomes during mitosis
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Sister chromatids structure
- kinetochores in middle
- cohesion proteins holding them together
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Mitosis phases
- G1 - primary growth phase, prep for DNA synthesis
- S - replication of DNA
- G2 - organelles replicate
- M - 5 phases, chromosome segregation into daughter cells
- C - separation of 2 new cells, division of cytoplasm
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Phase timings
- cell division takes approx 24 hours in mammalian cell
- mitosis takes approx 1 hour
- G1 has most variation in length
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Prophase
- chromatin condenses into chromosomes which appear as two sister chromatids held by centromere
- nuclear envelope breaks down
- spindle apparatus assemble
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Prometaphase
- nuclear membrane breaks apart
- chromosomes attach to microtubules at kinetochores
- chromosomes move to equator of cell
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Metaphase
- chromosomes align in middle of cell
- attached to opposite poles + under tension
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Anaphase
- cohesion proteins degraded
- chromosomes pulled to opposite poles as centromere splits
- spindle poles move apart
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Telophase
- chromosomes reach poles + decondense back into chromatin
- nucleomembrane forms around each set of chromosomes
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Cytokinesis
physical division of cytoplasm resulting in two daughter cells
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G1/S checkpoint
- cell decides to divide
- primary point for external influence
- use Cdc2/G1 cyclin
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G2/M checkpoint
- cell makes commitment to mitosis
- assesses success of DNA replication
- uses Cdc2/mitotic cyclin
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Late metaphase (spindle) checkpoint
- ensures chromosomes attached to spindle
- uses APC
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Cyclin-dependent kinases (Cdks)
- enzymes that drive cell cycle transition with proper progression
- phosphorylate proteins activate/inhibit proteins at checkpoints
- primary mechanism of cell cycle control
- partner with different cyclins at different points in cycle
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Cdks control in eukaryotes
- multiple Cdks to control cycle
- cells respond to greater variety of external signals
- more complex controls
47
Cancer
- uncontrolled cell division
- uncontrolled growth of cells
- failure of cell cycle control - failure in checkpoints, mutated genes
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Requirements for DNA synthesis
- pre-existing single stranded DNA template
- pre-existing free 3' hydroxyl group
- protein catalyst (DNA polymerase)
- dNTP precursors --> dATP, dCTP, dGTP, dTTP
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Polymerase Chain Reaction (PCR) definition
- mimics DNA replication to produce millions of copies of a DNA sequence
- allows amplification of small DNA fragments using primers that flank region
- DNA doubles each time = exponential amplification process
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Taq polymerase
heat-stable DNA polymerase enzyme, allows process to run efficiently at high temperatures
51
PCR steps
- denaturation - high temperature separates DNA double strands
- annealing of primers - low temperature, primers bind to complementary DNA sequence
- DNA synthesis (extension) - intermediate temperature, polymerase begins synthesis
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Reverse transcription PCR (rt-PCR)
- PCR performed on cDNA made from mRNA
- allows creation of recombinant DNA containing only exons of genes
- allows study of structure and function of gene products + relative levels of gene expression in cells
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Quantitative RT-PCR
- measures number of copies of DNA synthesized in real time using PCR machine --> from rt-PCR
- use of DNA-binding probes attached to template
- once template denaturized, fluorophore on probe fluoresces
- signal detected by machine once probe cleaved
- graph constructed of fluorescence unit vs cycle number
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Steps of rt-PCR
- isolation of mRNA
- conversion to cDNA using reverse transcription
- PCR amplifies specific cDNAs
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Uses of PCR
- analysis of sperm cells or hair --> amplify DNA to allow for analysis
- use primer selectivity to identify likelihood of individual carrying particular allele
- diagnosis of disease --> contain nucleic acids which can be identified by PCR, diagnosis kits in COVID
- DNA fingerprinting --> identify individual based on tissue/fluid using STRs, used for paternity, crime suspects (CODIS), conservation biology
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DNA sequencing definition and role
- determines exact order of nucleotides in DNA molecule
- used for studying genomes, identifying mutation + diagnosing disease
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Sanger method
- DNA sequencing with chain-terminating inhibitors, interrupted replication
- includes template DNA, labelled ddNTPs, primers, DNA polymerase
- mix of dNTPS and ddNTPS create variety of DNA fragments
- run electrophoresis to separate fragments
- high quality + long reads but low output
- sequence obtained is reverse + complement of template
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Sanger sequencing adv and disadv
- must construct genomic library
- need to prepare many reactions simultaneously but enables high quality long chains
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Next-Gen Sequencing
- increased speed
- reactions simultaneous
- short fragments sequenced in real time
- improvements in cost per sequence base
- no need to construct genomic library
- mass amounts of data
- produces have less information per molecule to ensure quality - as read length increases, quality decreases
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Challenges in genomics
- complexity, challenge to reconstruct + sequence genome
- polyploidy = organism that has more than 2 sets of chromosomes - more repetitive DNA, increased redundancy + complexity
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Human genome project
- 2003
- aim to map entire human genome by determining location of genes on chromosomes and understanding variation
- first draft took 11 years and cost $3 billion USD
- now around $600 and couple days to produce
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Challenges in genome sequencing
- large amounts of sequencing reads but location + orientation in genome unknown
- need to put together like a puzzle but with repetitive pieces
- challenging to get right position
- assembly process = challenge
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Parts to gene expression
- central dogma
- transcription
- translation
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Gene
segment of DNA that contains instructions for making specific protein/RNA
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One-gene/one-enzyme hypothesis
- genes specify structure of enzymes
- relationship between genotype and phenotype
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Central dogma
information flow from DNA to RNA to protein
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Transcription
- DNA directed synthesis of RNA
- nucleic acid language
- flow from DNA to RNA
- based on complementarity
68
Translation
- RNA synthesizes protein
- translate from nucleic acid language into protein language
69
Copied strand in transcription
template strand, antisense
70
Uncopied strand in transcription
coding strand, sense, mRNA identical to coding strand
71
mRNA
intermediate form of information from nucleus to cytoplasm for processing
72
tRNA
- intermediary adaptor molecule between mRNA and amino acids during protein synthesis
- acts as translator
- helps bring amino acids to ribosome
73
rRNA
essential for function in protein production, critical to ribosome
74
Small nuclear RNA
involved in processing of pre-mRNA in splicing
75
Signal recognition particle RNA
mediator for proteins synthesized on RER
76
Small RNAs (miRNA + siRNA)
gene expression
77
Codon
block of 3 nucleotides corresponding to an amino acid
78
Frameshift mutations and effect on codons
- add/del of 1/2 nts shifted genetic message and altered all aa after deletion
- add/del of 3 nts resulted in normal protein after add/del
- genetic code read in increments of 3 nts continuously
79
Codons that specify each amino acid
- stop codons: terminate translation, UAA, UGA, UAG
- start codons: signify start of translation, AUG, also codes for methionine
- 64 codons encoding 20 amino acids
- some amino acids specified by more than one codon
80
Number of RNA polymerases required in prokaryotic transcription
single RNA polymerase
81
Forms of RNA polymerase
- core polymerase: comprised of four subunits that hold protein together, cannot initiate synthesis accurately
- holoenzymes: formed by addition of sigma subunit to core polymerase, initiates synthesis as sigma has ability to recognize promoter sequences
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Transcription requirements
- promoter: forms recognition + binding site for RNA polymerase
- start site: actual site where RNA synthesis begins, +1 = first base
- terminator: signal to end transcription
- transcription unit: region from promoter to terminator
83
Promoter
upstream of start site, initiation occurs but not transcribed by polymerase, two signals - TATA box and TTGCA
84
RNA synthesis starting at +1
downstream of start site
85
Transcription initiation
- sigma subunit recognizes promoter elements at -35 and -10 and binds to DNA
- DNA helix unwinds at -10
- polymerase recognizes start and binding site based on sigma
- once polymerase knows correct orientation, sigma disassociates
- transcription begins at start site, +1
86
Transcription elongation
- ATP forms 5' end of chain
- RNA chain grows in 5'-to-3' direction as nucleotides added
- clearance = process of leaving promoter
- transcription bubble = RNA polymerase, DNA template + growing RNA transcript
- after transcription bubble passes, DNA rewound
87
Transcription termination
- terminator = G-C pairs which form double-stranded hairpin, followed by series of A-T base pairs
- structural change causes polymerase to pause
- the 2 H-bonds between A-T not sufficient enough to hold RNA-DNA hybrid
- mRNA disassociates from RNA
- polymerase releases DNA and DNA rewinds
88
Transcription in prokaryotes
- transcription + translation couple
- operons: single mRNA that contains multiple genes that are functionally related + regulated together
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Transcription in eukaryotes
- transcription + translation not coupled
- no operons
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Number of RNA polymerases required in eukaryotic transcription
- 3 different polymerases each with own promoter
- RNA pol I transcribes rRNA
- RNA pol II transcribes mRNA and some snRNA
- RNA pol III transcribes tRNA
91
Core promoter of RNA pol II
different elements including TATA box
92
Initiation of transcription in eukaryotes
- initiation of transcription at pol II promoters requires series of transcription factors
- RNA pol II associates with transcription factors + DNA forming initiation complex, transcription begins
- also require elongation complex factors + chromatin-remodeling complex
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pre-mRNA modifiations
- in nucleus
- addition of 5' cap for RNA stability in further processing
- addition of 3' poly-A tail
- removal of introns via splicing
94
Introns
intervening sequences, genes interrupted by sequences not in mRNA and proteins
95
Exons
expressed sequences, translation
96
Transcriptome
all RNA produced from genome
97
Proteome
all proteins produced from genome, size unknown
98
Ribosome structure and function
- two subunits: large and small
- consists of rRNA and proteins
- key macromolecule involved in translation
- multiple tRNA binding sites
- A site: tRNA carrying next aa to be added
- P site: tRNA attached to growing peptide chain
- E site: tRNA that carried previous aa
99
tRNA structure and function
- L shaped molecule with 2 ends, interact with mRNA and aa
- aminoacyl-tRNA synthetases add aa to acceptor end of tRNA - hydroxyl end attaches to carboxyl end of aa, charging reaction
- anticodon loop contains 3 nts complementary to mRNA codons
100
Translation initiation
- in cytoplasm
- mRNA threads through ribosome while initial tRNA carrying aa binds to ribosome
- subunits attach and elongation begins
- prokaryotes = special imitator tRNA with chemically modified methionine
- eukaryotes = methionine as initiating aa, more protein factors in initiation complex
101
Translation elongation
- aa added continuously
- starts with second tRNA binding to site
- peptide bond formed between aa of initiator tRNA at P-site and newly arrived charged tRNA at A-site
- ribosome moves 5'-3' and tRNA with growing chain of aa moves to P-site, next tRNA shifts into A-site, empty tRNA leaves through E-site
102
Wobble pairing
- flexibility through modified bases
- single tRNA = multiple codons
- allows less stringent pairing between codon and anticodon
103
Translation termination
- stop codon reached
- recognized by release factors
- release newly made polypeptide from ribosome
104
Translation on RER
- signal sequences at beginning of polypeptide sequence bind to signal recognition particle in cytoplasm to form complex
- complex recognized by RER receptor proteins
- ribosome binds to membrane = docking
- signal sequence in membrane proteins initiate translation on ER membrane
- protein passes through channel in ER membrane = protein-trafficking highway
- can be modified to be released outside cell
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Mutation definition
heritable change in genetic material
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Point mutation
single base change, leads to single nucleotide variation in populaltions
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Types of point mutations
- silent mutation - same aa, no net effect
- missense mutation - different aa, no effect or non-functional protein
- nonsense mutation - changed to stop codon, incomplete protein
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Indel mutation
insertion or deletion of 1-50 bp
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Frameshift mutation
addition/deletion of base, alter sequence of aa downstream
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Structural variation
mutations that affect >50 bp
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Types of structural variation
- copy number variation - difference in number of copies of particular genetic region, loss of DNA via deletion or gain of DNA via insertion/duplication
- inversions - entire chromosome segment inverted
- reciprocal translocation - chromosome segments transferred between chromosomes
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Mutation rate dependent on...
selection pressure
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Agrobacterium
- transfers DNA to plant genome causing growth of gall, Crown Gall disease
- modification to replace with gene of interest --> bacteria transfers gene to plant
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GM tomato
- FLAVR SAVR tomato
- first GM food crop
- took wall softening gene from tomato + cloned into expression vector backwards
- downregulated expression of gene to make firmer tomatoes
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CRISPR
- genome editing
- gene knockout - targets gene, cuts DNA and repair causes deletion
- gene editing - specifically modify bases using template DNA
- helped accelerate crop production, add traits, domesticate new crops
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Applications of biotech in food
- enviropig, reduction in water pollution
- aqua advantage salmon, growth hormone introduced
- myostatin, mutation that produces double muscle mass edited into cattle
- polled (hornless) gene in cattle embryo
- featherless chickens, for warm countries
- cloning of sheep from tissue cells
117
Application of biotech in medicine
- gene and stem cell therapy - restore defective genes, stem cells to treat degenerative disease and tissue damage
- take body cells and transform them back into stem cells to create new cell
- gene treatments for sickle cell anemia with CRISPR
- germline gene therapy - changing genes of embryo in IVF + embryo selection
