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What is a Cloning vector?

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1

What is a Cloning vector?

A stable replicating DNA molecule. Often bacterial DNA

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Restriction enzymes

  • Recognize specific sequences of dsDNA

  • Sticky and blunt ends

  • Occur naturally in bacteria. Will cut a DNA molecule wherever the recognition sequence occurs.

  • Engineered nucleases consist of DNA the binds to a chosen target sequence linked to a restriction enzyme → cut at a predetermined DNA sequence

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Sticky ends

Enzymes that cut to leave a short ssDNA end

  • Will spontaneously form dsDNA → DNA fragments can be glued together

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Blunt ends

Enzymes that cut the DNA with no ssDNA end

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Visualizing DNA

“Tag” DNA with chemical markers to make it visible

  1. UV-radiating reagents - general

  2. Radioactive probes - specific

  3. Flourescent markers - specific

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Dideoxy sequencing

  • Similar to PCR, but some differences:

    • Only 1 primer

    • More template

    • Small amounts of ddNTPs included (which label by colour)

  • When making the new complementary chain ddNTPs will be randomly selected among other nukleotides, ending that chain.

  • Run high resolution electrophoresis to separate by length.

  • Determine sequence from order of different colours (marking different ddNTPs)

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ddNTP

Like a regular nukleotide but nothing can bind after it, so it at the end of its chain.

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Next Generation Sequencing

  • Sätt visualisera DNA

  • How:

    • Cut genome with restriction enzyme and add unique adaptor sequende

    • Process

    • Each base has a different colour

    • The adaptor sequence identifies the source of the DNA

  • Cheeper and faster than dideoxy sequencing

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Thrid Generation Sequencing

  • Analyzing DNA molecules in real time

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CRISPR-Cas editing

  • Effector comlex associates with PAM (short & frequent). If seed sequence matches genome sequence clode to PAM → dsDNA/RNA → cutting by CAS9

  • Repair the cut:

    • NHEJ → insertions or deletions (knock out gene)

    • Homologous recombination with donor DNA (introduction of new DNA)

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Transgenic organism

Organism with foreign DNA introduced into genome

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Genetically Modified Organism

Organism with artificially modified DNA

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Effector complex

Used in CRISPR-Cas

  • Single guide RNA + CAS9 protein

  • The guide controls where it will cut

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Amplify DNA

  • Often you don’t have a lot of DNA to work with and have to amplify it

    • e.g. if DNA from crime scene or ancient DNA

  • Different techniques:

    • PCR

    • qPCR

    • Bacterial cloning

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qPCR

  • quantitative Polymerase Chain Reaction

  • Same process as PCR but the amplified DNA is flourescently labled

    • qPCR is also in real time so you can monitor the amplification as it’s happening

  • The amount of flourecence released during amplification is directly proportional to the amount of amplified DNA

  • Mäter genuttryck

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Varför jämför man ofta non-coding regions mellan människor?

  • Den del som mest troligt varierar mellan personer

    • Varierar i antal repetitioner

  • Hela genomen är alldeles för stor för att kunna undersökas

    • 99% av genomen är samma mellan olika individer

      • Vi jämför den procent som skiljer

        • Ex no-coding regions

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Packing

  • Makes the DNA inaccessible but can be done flexibly

  • Ex. supercoiling and chromatin

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Supercoiling

The coil of the DNA coiling around itself

  • Occurs when the molecule relieves the helical stress by twisting around itself

  • Regulates access to genetic code

  • Both eucaryotic and procaryotic

  • For procaryotes DNA is globally ______, but for eucaryotes it is done locally and correlated with transcription active regions

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Positive and Negative Supercoil

Positive:

  • In the direction of DNA helix (right), makes it tighter

  • Less common

Negative:

  • In the opposite direction of DNA helix (left), makes it looser

  • More common

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Topoisomerase

Enzyme that introduces or removes negative supercoils

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Chromatin

Wrapping DNA around histones which group to make chromatids

  • Histones are positively charged while DNA is negative

  • If loosely packed some genes are accessible

  • DNA methylization: inhibits gene expression

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Chromosome puff

Regions of relaxed chromatin

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Heterochromatin

Highly condensed, gene-poor and transcriptionally silent

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Euchromatin

Less condensed, gene-rich and more accessible to transcription

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Centromere

Small specialized chromosomal regions

  • Without them cells cannot divide properly

  • Very conserved but no specific sequence

  • Histones are possibly involved

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Telomere

Ends of chromosomes

  • Made of repetitive non.coding DNA

  • Shorten with time, when lost the cell dies

    • Predicts life span

  • “End replication problem”

  • Length is linked to early life adversity

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Semiconservative

The process of replication is __________, there is one original DNA molecule and one new copy

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Theta model

Procaryotic replication

The new copy is made in the middle and DNA synthesis happens at both sides of the fork

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Primase

Enzyme that synthesizes short RNA sequences called primers

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Nucleosome

A segment of DNA wound around eight histones

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End replication problem

Telomere length progressively shortens because of the inability of DNA polymerase to fully replicate the 3ʹ end of the DNA strand.

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Recombination

  • DNA molecules exchange pieces of genetic material

  • Happens during meiosis

  • Increases genetic variation

  • Essential for some types of repair

  • Give info about linkage of genes

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Transcription

  • The synthesis of an RNA molecule from a DNA template

    • RNA is transcribed from a single DNA strand/gener transkriberas alltid från antingen ena eller andra strängen

  • Controls gene-expression

  • Tre faser:

    • Initiation

    • Elongation

    • Termination

  • Syntetiseras av RNA-polymeras

    • No primer is needed

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Transcription unit

  • Promoter,

  • RNA coding region,

  • & terminator

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RNA polymerase i bakterier vs EU-celler

  • Bacterial cells possess only one type of RNA polymerase

    • catalyzes the synthesis of mRNA, rRNA and tRNA

  • Eukaryotic cells possess several types of RNA polymerase

    • Transkriberar olika sorters RNA

    • Fler saker att kontrollera i eukaryot cell

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Sigma factor

Procaryotic transcription initiator

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Initiation (transkription)

  • RNA polymerase binds to a promoter region

  • Requires sigma factor in procaryotes

  • Both a core and regulatory promoter in eucaryotes

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Holoenzyme

  • Sigma factor + RNA polymerase → RNA polymerase _____________

  • Binds and unwinds promoter NDA, forming the transcription bubble

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Intron

  • Non-coding regions of DNA

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Exon

  • Coding regions of DNA

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Pre-mRNA processing

  • Only in eucaryotic cells

  • Addition of a cap to the 5’ end

  • Stops it from being degraded

  • Addition of poly(A) tail at 3’ end

    • Skyddar från degradation

  • Splicing

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Consensus sequence

  • The places at the beginning and end of an intron where it is cut

  • Tells spliceosome where to cut

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Alternative splicing

Kan utöver att ta bort introner även ta bort exoner → nya kombinationer av den kodande informationen i genen → olika mRNA från samma del i DNA’t

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Multiple 3’ cleavage sites

  • Jämförbart med alternative splicing

  • Kan ta bort exoner i mRNA

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Bacterial CRISPR-Cas

  • Adaptive and inheritable immune system

    • Defense against invasive genetic elements (such as viral DNA )

  • Gene silencing in the lab using CRISPR:

    • Able to knock in genes, knock out genes or mutate sequences

    • Jämför med RNA interference som tillfälligt tystar gener by knocking down their mRNA transcripts

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Coding RNA

  • mRNA that encodes protein

    • (to act as various components including enzymes, cell structures, and signal transductors)

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Noncoding RNA

  • RNA that acts as cellular regulators

    • Without encoding protein

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Gene expression

  • The process by which the information encoded in a gene is used to direct the assembly of a protein molecule

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RNA polymerase holoenzyme

  • I prokaryotiska celler

  • RNA polymerase och sigma factor (σ factor)

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RNA polymerase I

  • Eukaryota celler

  • Transkriberar rRNA

  • I nukleolen

    • Där ribosomer bildas

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RNA polymeras II

  • Syntetiserar pre-mRNA

    • pre-mRNA = all mRNA innan den är helt mature and ready to be translated

  • I cellkärnan

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RNA polymeras III

  • Eukaryota celler

  • Transkriberar ex pre-tRNA och snRNA

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snRNA

  • Small nuclear RNA

  • have a variety of functions, including:

    • “splicing” pre-mRNAs

    • regulating transcription factors.

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Prokaryotisk cell vs eukaryotisk - DNA

  • Prokaryotisk:

    • DNA is smaller and more simple

    • DNA is also circulat

  • Eukaryotisk:

    • DNA is linear

  • Base pairs are the same but the replication process differs

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How does the DNA of mithochondria and chloroplasts differ from that found in the nucleus?

  • High copy number

    • A mitochondrion or chloroplast has multiple copies of its DNA,

  • Random segregation

    • Mitochondria and chloroplasts (and the genes they carry) are randomly distributed to daughter cells during mitosis and meiosis.

  • Single-parent inheritance

    • Non-nuclear DNA is often inherited uniparentally, meaning that offspring get DNA only from the male or the female parent

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Which replication ways were first proposed? Which is true? (Kopplat till hur nytt och gammalt DNA fördelas)

  • Conservative

    • Both old DNA strands go to one cell, both new ones to the other

  • Semiconservative

    • True, what really happens

    • A new strand is added to each of old strands

  • Dispersive

    • Parts of old and new strands are mixed in both daugther cells

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Which are the modes of replication?

Ways DNA can replicate

  • Theta model

  • Rolling-circle model

  • Linear eucaryotic replication

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Rolling-circle model

  • Mode of replication

  • Replication along one sigle strand

  • DNA synthesis begins at 3’ end of the broken nucleotide strand

  • The 5’ end is continuously displaced as the DNA cell unwinds

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Replication bubble

  • The chromosomes in eucaryotic cells contain several origins of replication

  • Replication happens at the origins at the same time

  • Replication bubbles are created when the DNA is unwound at each origin of replication

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Differences eucaryotic DNA and bacterial DNA when it comes to replication

  • DNA in EU-cells is much longer→ replication is initiated at multiple origins

  • EU -cells have linear chromosomes, bacteria circular

  • Nucleosome assembly must immediately follow DNA replication in EU-cells

    • Wrapping around histones

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Gene conversion

  • Specific type of homologous recombination

  • Involves the unidirectional transfer of genetic material from a 'donor' sequence to a highly homologous 'acceptor'

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Promoter region

Sequence of DNA that starts RNA transcription

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Secondary structure in RNA

Created by RNA folding

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Elongation

  • Only one side of DNA is the template

    • The RNA transcript carries the same info as the non-template (coding) strand of DNA, but has U insted of T

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What is NTPs?

A, C, G, U, building blocks of RNA

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Terminator site

Section of DNA that marks the end of a gene during transcription

  • Causes transcript to be released

  • Series of UUUU… slows transcription which causes hairpin loop of complementary RNA

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mRNA

Template for protein synthesis

  • Carries info from gene to ribosome

  • Removes the introns in DNA, done by splicing

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Splicing

Removal of introns

  • Takes place in the spliceosome

  • Requires consensus sequences

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rRNA

  • 2 units, one big and one small

  • Keeps mRNA stable and makes it clear where tRNA goes

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