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What is a Cloning vector?

What is a Cloning vector?

A stable replicating DNA molecule. Often bacterial DNA

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

Sticky ends

Enzymes that cut to leave a short ssDNA end

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

Blunt ends

Enzymes that cut the DNA with no ssDNA end

Visualizing DNA

“Tag” DNA with chemical markers to make it visible

1. UV-radiating reagents - general

2. Radioactive probes - specific

3. Flourescent markers - specific

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)

ddNTP

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

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

Thrid Generation Sequencing

• Analyzing DNA molecules in real time

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)

Transgenic organism

Organism with foreign DNA introduced into genome

Genetically Modified Organism

Organism with artificially modified DNA

Effector complex

Used in CRISPR-Cas

• Single guide RNA + CAS9 protein

• The guide controls where it will cut

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

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

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

Packing

• Makes the DNA inaccessible but can be done flexibly

• Ex. supercoiling and chromatin

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

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

Topoisomerase

Enzyme that introduces or removes negative supercoils

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

Chromosome puff

Regions of relaxed chromatin

Heterochromatin

Highly condensed, gene-poor and transcriptionally silent

Euchromatin

Less condensed, gene-rich and more accessible to transcription

Centromere

Small specialized chromosomal regions

• Without them cells cannot divide properly

• Very conserved but no specific sequence

• Histones are possibly involved

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

Semiconservative

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

Theta model

Procaryotic replication

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

Primase

Enzyme that synthesizes short RNA sequences called primers

Nucleosome

A segment of DNA wound around eight histones

End replication problem

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

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

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

Transcription unit

• Promoter,

• RNA coding region,

• & terminator

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

Sigma factor

Procaryotic transcription initiator

Initiation (transkription)

• RNA polymerase binds to a promoter region

• Requires sigma factor in procaryotes

• Both a core and regulatory promoter in eucaryotes

Holoenzyme

• Sigma factor + RNA polymerase → RNA polymerase _____________

• Binds and unwinds promoter NDA, forming the transcription bubble

Intron

• Non-coding regions of DNA

Exon

• Coding regions of DNA

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

Consensus sequence

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

• Tells spliceosome where to cut

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

Multiple 3’ cleavage sites

• Jämförbart med alternative splicing

• Kan ta bort exoner i mRNA

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

Coding RNA

• mRNA that encodes protein

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

Noncoding RNA

• RNA that acts as cellular regulators

  • Without encoding protein

Gene expression

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

RNA polymerase holoenzyme

• I prokaryotiska celler

• RNA polymerase och sigma factor (σ factor)

RNA polymerase I

• Eukaryota celler

• Transkriberar rRNA

• I nukleolen

  • Där ribosomer bildas

RNA polymeras II

• Syntetiserar pre-mRNA

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

• I cellkärnan

RNA polymeras III

• Eukaryota celler

• Transkriberar ex pre-tRNA och snRNA

snRNA

• Small nuclear RNA

• have a variety of functions, including:

  • “splicing” pre-mRNAs

  • regulating transcription factors.

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

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

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

Which are the modes of replication?

Ways DNA can replicate

• Theta model

• Rolling-circle model

• Linear eucaryotic replication

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

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

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

Gene conversion

• Specific type of homologous recombination

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

Promoter region

Sequence of DNA that starts RNA transcription

Secondary structure in RNA

Created by RNA folding

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

What is NTPs?

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

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

mRNA

Template for protein synthesis

• Carries info from gene to ribosome

• Removes the introns in DNA, done by splicing

Splicing

Removal of introns

• Takes place in the spliceosome

• Requires consensus sequences

rRNA

• 2 units, one big and one small

• Keeps mRNA stable and makes it clear where tRNA goes