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203 Terms


Bianary Fission

  • A form of asexual reproduction

  • 1 parent cell → 2 identical daughter cells

  • make exact copies of genome, one to each dauughter cell

  • Prokaryiotic cell division = reproduction

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Process of Binary Fission

Includes iniation, elongation and termination

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  • The DNA of chromosome attached by a protein to plasma membrane

  • begins along the origin of replication region of chromosome

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  • Newly synthesized DNA attached to plasma membrane

  • Cell elongates until 2 DNA attachment sites are on opposite sides of the cell (which is double its size)

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  • Cells start to contract along the middle until the new cell wall and membrane is reformed creating 2 identical daughter cells

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Eurkaryotic cell division - mitosis

  • occurs. only in stomatic cels

  • allows unicellular fertilized eggs to develop into multicellular organisms

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Stem cells

  • eukaryotic cell division - mitosis

  • are unspecialized and embryonic ones can reproduce indefinitely under appropriate conditions and differentiate into more than one type od specialized cells

  • adult stem cellscan only replace non-reproductive specialized cells

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Eukaryotic cell division - mitosis example

Adult musscle tissue

  • adult muscle tissue is stable with little cell division (turnover)

  • if injured, non dividing (quiescent) satellite stem cells in basement membrane of tissue are activated and start to divide causing muscle regeneration (proliferation)

  • Differentiation + fusion → muscle precursor cells (myoblasts - eventually form myofibers/muscle cells)

  • Myofibers are unable to divide once formed

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Process of Mitosis

  • Eukaryotic DNA is larger and more organized into linear chromosomes and highly condensed in the nucleus

  • 2 distinct stages: interphase and the M phase

    • Interphase: S (synthesis) phase (DNA synthesis), gap growth phases (G1 and G2) o M phase: mitosis and cytokinesis

  • 1 mitotic cell division = chromosome replicated and separated into daughter cells

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Cells prepare to divide (replicate DNA in nucleus + increase of cell size), chromosomes in long thin chromatin fibers

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S Phase

Replication of DNA occurs (end to end)

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G1 and G2

Prepare cell for DNA synthesis then mitosis

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Organelles used to make protein machinery are built

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G1 and G2 role

Ensures parent cell is large enough + has the organelles needed for mitosis + has normal functions

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G0 phase

Pause of the cell cycle between M and S phase (long or short depending on cell type)

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Cells permanently in G0 phase are non-dividing

eye lens cells, nerve cells, mature muscle cells

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Chromosomes must be duplicated and condensed before entering mitosis = sister chromatids (23 distinct pairs)

idk what to write on theis side lol just know that :)

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Example of the process of mitosis - stem cells

  • reproduce indefinitely and also have periods of quiescence (no cell division)

  • Fully differentiated skeletal muscle has little to no cell division; upon injury, quiescent satellite stem

    cells are activated from dormant G0 phase and re-enter cell cycle

  • Enables proliferation, differentiation, and maturation of new muscle fibers

  • Once myofibers formed, they exit cell cycle, enter G0 phase

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5 stages of mitosis by Walther Flemming

Prophase, Prometaphase, Metaphase, Anaphase, Telophase, Cytokinesis

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  • 4 chromosomes 4 centromeres 8 chromatids

  • Still in the nuclear envelope, each chromosome = identical sister chromatid joined with centromeres (microtubule organizing centers). Centromeres radiate long microtubules forming mitotic spindle fibres. Chromosomes condense, and centrosomes move to opposite poles

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  • 4 chromosomes, 4 centromeres, 8 chromatids

  • Fragmentation of nuclear envelope from breaking down (allow spindles to attach to a region on centromeres and kinetochores:

    • Specialized protein structures that match to one chromatid

  • Some microtubules that radiate from the centrosome attach directly to kinetochore regions

    • Pull chromosomes to polar sides of the cell or push poles away from each other

  • Some also radiate from centrosomes as part of the mitotic spindle and are polar

    • Interact w/ each other to help push poles oppositely

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  • 4 chromosomes, 4 centromeres, 8 chromatids

  • Alignment of chromosome at center of cell (metaphase plate) Kinetochore microtubules attached at kinetochores facilitate this movement

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  • 8 chromosomes, 8 centromeres, 0 chromatids

  • Kinetochore microtubules shorten and sister chromatids separate into individual chromosomes that are pulled towards poles + polar microtubules push against each other to help elongate the cell. Ends of the cell have equal and complete chromosome sets

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  • 8 chromosomes

  • 2 new daughter nuclei form and nuclear envelope reforms around chromosome at opposite poles of the dividing cell. Chromosome decondenses and spindle microtubules are depolymerized (broken down).

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  • 4 chromosomes split apart

  • Division of the cytoplasm of cell

    • Animals: forms contractile ring of motor proteins that contract actin fibre bundles along midline giving defined cleavage making 2 cells, membrane reforms

    • Plants: have a cell wall, lay down new cell wall along plate region in the middle of dividing cell, wall fuses with original wall making 2 cells

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Controlling progression (tim hunt) - procedure

measured protein level changes of dividing sea urchin embryos, + radioactively labelled aa (methionine) to eggs, took samples of dividing embryos every 10 min, used gel electrophoresis

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Controlling progression (tim hunt) - findings

most protein bands on the gel became darker as cell division and embryonic development progressed. One protein band oscillated in intensity, protein increased then decreased with each subsequent division. Cyclical nature therefore named it cyclin

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Controlling progression (tim hunt) - follow up work

mitosis promoting factor = cyclin protein and cyclin-dependent kinase (CDK) protein that controlled the progression of the cell cycle

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proteins that oscillate in concentration according to the cell cycle and bind with a cyclin dependent kinase (CDK) to control cell cycle

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add phosphate groups (phosphorylation) to activate or deactivate amino acids on target proteins; activity dependent on bind to cyclin

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protein kinase, uses ATP to add phosphate groups to protein that induces conformational change to be active, it must be attached to cyclin

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Cyclin-CDK complex triggers the changes during various cell division cycles, specifically phosphorylation of target proteins that promote division

i have nothing to write here so just know that

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G1/S cyclin – CDK Complex:

Helps transition from G1 to S phase as it prepares cell for DNA replication by increasing the expression of histone proteins \n

(uses cyclin to regulate cell cycles)

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S – Cyclin – CDK Comple

Helps initiates DNA synthesis \n

(uses cyclin to regulate cell cycles)

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M – Cyclin – CDK Complex

Initiate mitosis (ex: facilitated by phosphorylation of proteins are needed for the nuclear membrane to break down and regulate the assembly of microtubules in mitotic spindles)

(uses cyclin to regulate cell cycles)

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3 Major checkpoints

“Cell watch”, block cyclin-CDK activity if mishap pauses cell division until the next phase is ready

  • DNA Damage Checkpoint (end of G1 before S phase)

  • DNA Replication Checkpoint (end of G2 phase)

  • Spindle Assembly Checkpoint (in M phase before anaphase)

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DNA Damage Checkpoint (end of G1 before S phase)

  • Only undamaged DNA enter S phase for replication,

  • Strand or backbone breaks (phosphodiester bond) kinases phosphorylate P53

  • P53: protein that inhibits cell cycle when turned on (normally present at low levels in nucleus)

  • Once phosphorylated, P53 accumulates in the nucleus and can turn off genes that inhibit cell cycle leading to production of CDK inhibitor protein that binds and blocks G1-S cyclin-CDK complex activity to then pause the cell cycle in G1 phase giving cell time to repair damage

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DNA Replication Checkpoint (end of G2 phase)

When all DNA is replicated, and enters mitosis via the G2 checkpoint, the cell will only complete mitosis if all chromosomes are attached to a microtubule from the mitotic spindle due to the M phase checkpoint

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Spindle Assembly Checkpoint (in M phase before anaphase)

At prometaphase, regulatory proteins needed for spindle checkpoint monitor degree of attachment of sister chromatids to microtubules of spindles in kinetochore region

  • Unattached kinetochore produces a ‘wait’ signal that brings more regulatory proteins which are activated by the lack of tension in centromere

  • Only allow metaphase progression and anaphase entry when each sister chromatid binds to kinetochore microtubule, checkpoint proteins leave centromere, separase breaks chromatid bond

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a helical structure of purines pair with pyrimidines and hydrogen bonds between bases giving stability

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Semiconservative Replication (Watson and Crick)

  1. Hydrogen bonds are broken between strands so it starts to unwind

  2. DNA is copied, and each strand acts as a template to bring in new complementary strands

  • Each parent stand is a template for the daughter strand

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Support for Semiconservative Model (Meselson and Stahl)

Prokaryotic and eukaryotic

  1. Grow E. coli with radio isotope 15 N (mutate it) - Generation 0

  2. Transfer cells to medium with 14 N mutation and let cells divide one-time Generation 1

  3. Let cells divide 2nd time and compare DNA bands in a centrifuge Generation 2

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transition of information - Results

  • The centrifuge separates heavier DNA (15 N is the heaviest)

  • Gen 0 was purely 15 N

  • Gen 1 was 2 hybrids with one stand of 15 N and 14 N

  • Gen 2 had 2 hybrids, 1 pure 15 N and 1 pure 15 N

  • Starts in the S phase of cycle at origin of replication

  • DNA is copied from 3’- 5’ which produces a stand in 5’ to 3’

  • Incoming complementary nucleotide h bonds w/ template strand base

    pair + interacts with 3’ OH of polymer forming on daughter stand

  • Phosphodiester bond b/w growing daughter strand and new

    nucleotide to make backbone and pyrophosphate

  • Prokaryotes: begins at single origin where replication is continuous

    around the circular chromosome from initiation site

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Origins of replication

  • Origin sites are the specific points where DNA replication begins.

  • - DNA double helix opens up at the origin to form two single strands - Replication forks spread in both directions from the origin sites

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In DNA replication:

Both stands run anti parallel therefore they can both serve as template strands at the same time DNA ALWAYS READ 3’ TO 5’ \n mRNA ALWAYS FORMED 5’ TO 3’ \n ELONGATION LIMITED TO 5’ TO 3’

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Process of DNA replication

Initiation, primer binding, elongation, termination, proofreading

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DNA helicase unwinds double helix by breaking hydrogen bonds and separates strand into replication fork within the origin of replication

  1. Leading strand is in the 3’-5’ direction, 1 primer needed, continuous, replicated towards fork

  2. Lagging strand is in 5’-3’ direction, many primers needed, fragmented, replicates away from fork

  3. Strands tend to unwind; single-stranded binding proteins bind and stabilize parent strand until elongation

  4. Topoisomerases bind upstream of fork to reduce torsional strain from unwinding; serve as initiator proteins that trigger unwinding of replication origin; primer synthesis then follows

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Primer Binding

Primer needed because elongation can only be done from existing piece of RNA or DNA. Leading strand is simplest to replicate because a primer binds to the 3' end of strand, always replicating 3’ to 5’

  1. RNA primase lays down primers (starting points for replication)

  2. Where DNA III polymerase elongates from

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DNA polymerase catalyzes the polymerization of replicated daughter strands 5’ to 3’ and synthesizes a replicated DNA strand from primers annealed to template strand continuously (at the site of the primer)

  1. DNA polymerase only adds 3’ to 5’ direction, and lagging strand runs 5’ to 3’, many primers are needed

  2. Lagging stand begins replication by binding with multiple primers. Each primer is only serval bases

    apart so DNA polymerase adds Okazaki fragments in between primers, then another DNA polymerase replaces RNA primers with DNA nucleotide bases

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Leading and lagging strands formed, DNA polymerase I replaces RNA primers with appropriate bases, enzymes proofread. DNA ligase joins okazaki fragments together to form one stand by catalyzing a phosphodiester bond

a. Replacement of RNA primers with DNA nucleotides leaves a sugar phosphate backbone at the 3’ end with a free phosphate backbone, DNA ligase joins 3’ fragment end to adjacent DNA nucleotide via catalyzation of phosphodiester bond along backbone, thus joining fragments

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During replication DNA polymerases proof read each added base pair, if error is detected enzyme replaces the incorrect base pair and replication continues

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Shortening Problem

  • No way to complete replication at the 5’ end of daughter strands when DNA is linear

  • RNA primer on lagging strand is bound to very end of template strand, once RNA primer is removed, it

    cannot be replaced with nucleotides b/c there is no 3’ end available for nucleotide addition and

    phosphodiester bond formation = shorter and shorter strands with each replication

  • At end of parent strands are repeated DNA sequences, telomeres that protect the ends of chromosomes

  • Every time a cell replicates, the lagging strand gets a little shorter (loss of bases) therefore what gets lost

    of the telomeres, stress and age increases its shortening, when it runs out cell dies

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several repeated non-coding sequences of TTAGGG to act as gene buffer

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a specific reverse transcriptase that synthesizes the telomeres, in gametes and stem cells to ensure lifelong cell divisions with no genetic info lost (very active in cancer cells, rapid divide)

  • Binds to tail of telomere and catalyzes extension of template strand by adding telomere repeats. Once template strand is extended, primase, DNA polymerase and ligase go back to complete daughter strand replication for remainder of template strand

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  1. Using fluorescent labels to make the metaphase chromosomes blue and microtubules yellow

  2. At the start of anaphase, photo bleach on a section of microtubules is taken, this makes a small cleavage

    in microtubule that can be traced

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  • Original: Photo bleached section will still be visible as chromosomes begin to move

  • Altered: Photo bleached section will disappear when chromosomes begin to move

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  • Prediction proven right

  • Photo bleached section stayed visible, distance b/w chromosomes and photobleached section lessened

  • Assumed that the photobleached section would move and still be visible, proven right this means that

    the distance between the chromosome and damaged section shortens (only at one end)

  • The can move by releasing motor proteins

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Microtubules shorten at one end at the kinetochore

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Johnson & Rao

  • Cell fusion setup: Fuse M-phase cell with cells in G1, S or G2

  • Prediction: interphase cells will begin M phase

  • Null hypothesis: interphase cells will not begin M phase

  • Results: chromosomes in interphase cell condense signalling start of M phase

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Market & Masui

  • Microinjection setup: Inject cytoplasm from an M-phase cell into one frog oocyte, in another frog oocyte

    cytoplasm from interphase cell was injected

  • Prediction: one or both of the frog oocytes will begin M phase

  • Null hypothesis: neither of the frog oocytes will begin M phase

  • Results: Oocyte is driven into M phase (early mitotic spindle appears); oocyte remains in G2 phase

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M phase cytoplasm contains a regulatory molecule that induces M phase in interphase cells

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#1 cause of death in Canada. 1 in 2 Canadians will develop cancer in their lifetime. 1 in 4 Canadian will die of cancer. 5-year cancer survival ~ 63%. mortality rate declining since 1983. Incidence rate stabilizing. 82 000 Canadians will die of cancer in 2019. 220 400 Canadians will be diagnosed with cancer by 2019

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Normal cell

Nuclei - small and round

Arraignment - not many cells and organized pattern

Apperance- common size and shape (round/rigid, visible features)

Growth - normal cells will stop growth

Anchorage dependence: cells anchor to dish surface and divide Density-dependant inhibition: when cells have formed a complete single layer, they stop dividing \n Density-dependent inhibition: if some cells are scraped away, remaining divide to fill gap and then once they contact each other

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Cancer cell

Nuclei- large and odly shaped

Arraignnment - many dividing cells with a disorganized layout

Appearance - variation of size/shape, loss of normal features

Growth - continue to divide well beyond one layer forming clumps and overlaying, no anchorage or density dependant inhibition

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Cancer characteristics

  • Cycle is not regulated/controlled, cycle checkpoints are disrupted, and division is much faster

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cancer stages

  • Stage 1: tumor is small, not grown outside or origin organ

  • Stage 2: tumor is larger but has not spread

  • Stage 3: tumor is large and has spread to tissue and lymph nodes

  • Stage 4: tumor is spread through blood and lymphatic system to distant sites in the body

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Vincristine (from rosy periwinkle) and taxol (from pacific yew tree’s bark) are anti-microtubule agents. These plant extracts inhibit spindle fiber information resulting in disruption of mitosis

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Grew cancer research. Pim 1 biomarker for pancreatic cancer, afinitor drug, pancreatic cancer immunotherapy

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Cart therapy

Harnesses the potential of patients’ immune system to target and destroy cancer cells. They get T cells (type of lymphocyte that plays a role in immune response) and make them into CART cells in the lab which then is injected into the patient.

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  • This is the ability to amplify DNA in a tube via polymerase chain reaction (PCR) - Mary Mullis

  • Allowed for amplification (copying) of millions of copies of DNA from small starting sample

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  1. DNA is placed in a buffer with specific ions and salts and 15-30 long single strand DNA primers ○ Primers bind complementary to template strands and starting point for copying

  2. Deoxyribonucleotides (dNTPs) are added to aid the replication process

  3. To unwind, tubes are put in a thermocycler that goes through heat and cooling that promotes replication

  4. Taq polymerase is highly heat resistant and catalyzes the polymerization of each daughter strand

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Stages of PCR Reaction

Denaturation, annealing, extension, amplification

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Helix is unwound by heat from the thermocycler

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When mixture cools down the primers can anneal to their complementary DNA strands, primer s bind on opposite strands at each end of the target sequence

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DNA taq polymerase extends and polymerizes daughter stands using 4 dNTPs starting from primers and extending daughter stand 5’ to 3’

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each cycle doubles the number of DNA as primers bond to each copy too (2 n)

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Gel electrophoresis

Used to separate DNA fragments from other sources and PCR and used to separate macromolecules including RNA and proteins based on rate of movement through agarose gel in electric field

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DNA in Gel electrophpresis

  • Molecules put in well of porous gel and travel through it with electrical field applied to gel (+ and – end)

  • DNA and RNA are (-) (ionized P on phosphodiester bond) thus attracted to (+) anode end of gel

  • Fastest molecules moving through gel are smaller and slower molecules tend to be larger

    • This helps with particle separation

  • Standardized ladder (with fragments of known sizes) places in a well also

  • Utilizing fluorescent dyes, sizes of the band and therefore molecule sizes can be found

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DNA sequencing

Identifying the order bases appear in a DNA Sequence. This helps determine the function of gene, codes for proteins and non-coding regions

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  • Dideoxy chain-termination method

  • DNA to be sequenced used as DNA synthesis template, all key components in DNA replication needed

    • Denatured single-stranded template DNA, DNA primers (bind complementarily), free deoxyribonucleotides (dNTPs), DNA polymerase

  • B/c DNA polymerase catalyzes covalent bonds b/w 5’ P on nucleotide + 3’ OH on previous nucleotide, basis of dieoxy method needs modified deoxyribonucleotides (dNTPs)

  • Sanger: ddNTPs missing the OH at 3’ would not allow for further elongation of growing DNA strand b/c OH needed for attachment to next nucleotide

    • Labelled ddNTPs into sequencing rxn tubes + all other components needed for DNA replication

    • Based on this: ddNTPs in tube will lead to series of interrupted daughter stands, each termination at the site where ddNTPs incorporated

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  • Different fluorescent dye labelling each of the 4 ddNTPs to distinguish all chain terminators present

  • Same materials needed for PCR but also a dideoxy nucleotides for each of the 4 bases

  • Lack a OH on the 3' end of sugar ring (usually hydroxyl group acts as a hook to add bases to chain)

  • Once a dideoxy has been added to chain it terminates sequence as no bases can be added. Each chain

    end with a dideoxy that is dyed a color depending on its base

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  • Target DNA is copied many times making fragments of different lengths

  • Fluorescent chain terminator nucleotides mark ends of fragments which can determine sequence

  • Limitation: only determine a sequence of small fragments of DNA → shut gun sequencing was better

  • Mixture goes past PCR; DNA polymerase continue to add nucleotides until it happens to add a dideoxy one. After cycles, fragments different lengths, each have a fluorescent marker that shows a certain base

  • Will then go through gel electrophoresis to indicate the order of lengths each differing by one base and

    sequence of bases can be determined

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Shot gun sequencing - DNA fragments (myers and weber)

  • Multiple DNA sequencing assembled by examining regions of overlap between random DNA fragments

  • Based on being able to break entire genome into different sized pieces to then proceed with 3 phases

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Shot gun sequencing - computational sequencing

  • Critical to process of whole-genome sequencing, software capable of facilitating assembly of fragments

  • First bacterial whole genome sequenced: Haemophilus influenza (1998) → Humans (2000)

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Shot gun sequencing - phase 1: sequence

  • Random sequencing of DNA in each fragment

  • Normal ddNTP has OH on 3’C allowing this end to be elongated

  • ddNTP lacks 3’ OH, can’t be elongated b/c no OH to attack incoming nucleotide triphosphate

    • DNA polymerase can’t add another nucleotide to growing DNA strand once ddNTP is added

  • Replication stops at chain terminator

  • Sequence of template strand is unknown, elongation of daughter stand stops when ddNTP terminator

    with fluorescent tag is incorporated at 3’ end

    • Each of the possible ddNTPs is labelled with a different fluorescent dye

  • After interrupted daughter strands are separated by size, a trace of fluorescent tags show successive ddNTPs that terminate stand elongation + allow template sequence to be deduced

  • Once daughter strand sequence is known, sequence of template strand is determined

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Shot gun sequencing- phase 2: assembly

  • Based on sequencing similarities, info in fragments and identifying contigs b/w fragments

  • Identifying the regions of overlap between the generated fragments and inferring/assembling the

    long/continuous sequence of nucleotides in the DNA molecule that make up each chromosome

  • Regions of overlap called contigs are identified and then “put” together in a computer program to make 1 long continuous strand of nucleotides ATCG + CATCH = ATTACH

  • This allows for larger portions of chromosomes to be obtained based on sequence similarities

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Shot gun sequencing - phase 3 : annotation

  • Finding locations of genes, coding/non-coding regions in a genome, regulatory regions, + their functions

  • Get better understanding of A, C, G, T

  • Not all DNA in our genome is transcribed into RNA + not all RNA is translated into functional proteins

  • 6 reading frames for each double stranded DNA fragment

  • Establish correct reading frame by using computer program to scan genome in both

    directions and identify reading frame that works for both strand

    • Usually a long stretch of codons without a stop codon is a good indication s

  • Only one reading frame codes for the protein

    • Ex: human beta globin gene, only 1 reading frame will give full length protein, b/c other reading frames will terminate translation after only a few amino acids

  • Computers also look for start, typical codes for promoters and other regulatory sites (Eukaryote is trickier to identify introns and exons)

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In sequencing, annotation also requires the identification of patterns

Protein- coding regions, using hypothetical RNA, non-coding regions

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Protein-coding regions

Nucleotides that relate to amino-acids with no stop codons for stretches

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Using Hypothetical RNA

To infer DNA sequences (ex: tRNA has a hairpin loop that folds back onto itself which means it can be inferred to have complementary stretches) \n

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Non-Coding regions \n

Regions that code for non-coding RNA this helps with diversity in organisms

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How common are mistakes (dna mutations).

Mutations considered a source of genetic differences that either have devastating effects or beneficial adoption

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Causes of DNA mutations

Environmental factors or more likely DNA replication errors (can lead to propagation)

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Commonality of DNA mutations

In multicellular organisms they have a lower probability, But no unicellular

Ex: viruses (RNA virus has fastest mutation rate) b/c delicate nature of their RNA backbone, no proofreading

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Somatic mutations

  • Cell will be progenitor of population of identical daughter cells following cell division

  • but is not passed down to offspring. Early mutations lead to more widespread mutation in body.

  • If mutation arises in cell no longer dividing (post-mitotic in G0 cycle), mutations effect negligible

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Germline Mutations

Mutations that can be passed down to offspring. Germ cells are cells that come together to produce offspring like embryo

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Randomness of mutations

This was proven by Lederberg by using mutation caused by antibiotic resistance that were random


  1. Non-selective Plate 1- is any bacteria that grows on it

  2. Selective- bacteria from plate 1 was placed onto a second plate which was supplemented with penicillin.

    Some bacteria lived

    • The mutant culture of antibiotic-resistant was present prior to being exposed

    • Proved that genetic variations are random and that the environment did not create the mutation

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DNA repair

Mutations happen often, usually caused by mutagens (radiation and chemicals)

  • Specialized repair enzymes can fix these damages

    • DNA ligase:e can repair DNA backbone and single strand breaks

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Types of DNA repair - mismatch repair

  • Rreplication errors

  • DNA polymerase proofreading corrects.. If that doesn’t catch it, some repair enzymes recognize the mistake because a mismatched pair create s kink in the strand

    • Nnuclease enzyme cuts single strand, enzymes like DNA polymerase and DNA ligase close gap

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Types of DNA repiar - base excision repair

X-rays/Oxygen Radicals can cause a uracil to appear in DNA (uracil is only found in RNA)

  1. Uracil in DNA signals repair process to DNA uracil glycosylase

  2. DNA uracil glycosylase cleaves uracil from the deoxyribose sugar backbone, leaves behind bare deoxyribose sugar with no attached nitrogenous base

  3. AP endonuclease celas detects lack of base, the backbonecleaves both sides of backbone, leaves a gap for new base

  4. Other enzymesDNA synthesis occurs to close gap via DNA ligase and DNA polymerase (uses the intact stand as a template

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