Gene technology & Molecular Diagnostics
Given by Prof. Tina Kyndt | Els Van Damme | Marjan De Mey
Chapter I
Introduction to the basics (1)
1. Introduction
1.1. Genome
Genome = complete set of genetic material in a cell/organism
Can be analysed in it’s whole by whom-genome sequencing
Functional genome analyses needed to unravel the role of genes within those genomes
Transcriptome analyses to study the expression of certain genes or gene sets
o Transcriptome = embraces all RNA-molecules in cell or population, coding as non-coding
Proteom analyses to find out which proteins are synthesized
o Proteom = full collection of proteins that come to expression in a cel/tissue or specific circumstanses
Interactome analyses to discover how all these molecular interactions are linked
o Interactome = total of macromolecular interactions inside a cel or organism, mainly protein-protein and nucleic
acids-protein interactions
Library = DNA/RNA fragments that you physically store (-80°C) like that or first cloned in E. coli and then stores
Genomic library = contains DNA fragments of an entire genome of an organism → start whole genome sequencing or genome projects
Prokaryotic genomes
The bacterial “chromosome”
▪ Dubble stranded DNA in one single circular structure
▪ Lies in the nucleoid region = chromosome is compacted there
▪ Physical appearance is controlled by supercoiling ad loopforming
o So it becomes accessible for transcription and replication
o So its fits in cells (1 by 2 µm f.e.)
▪ Example E. coli
o 4600 kbp
o 4300 genes → 93% coding genes
o 7% non coding genes → no function or other functions like in the replication (ori for example) and DNA folding
▪ Ori = Origin of Replication = specific DNA sequence where the process of DNA replication begins
On a bacterial chromosome → oriC
o Master switch for the cell’s reproduction, it ensures the chromosome is copied exactly
once before the cell divides
o Usually just one oriC per bacterial chromosome
On a plasmid → oriV or ori
o On plasmids, the ori defines the plasmid’s personality → it determines how many copies
of it can exist (copy number) and which other plasmids can live with it (incompatibility)
o The gene order of E. coli resembles the gene order found in evolutionary related bacteria like Salmonella spp.
▪ Similar gene order on chromosomes of different species = synteny
E. coli and Bacillus subtills that are evolutionary more distinct
,Plasmids
▪ None to some present (copies or compatible different plasmids) in bacteria next to the larger genomic chromosome
▪ Possess their own ori → can replicate independently from chromosome
▪ Sometimes integrated in the chromosome = episome → plasmid replicates together with chromosome
▪ Plasmids can be grouped in incompatibility groups
o Plasmids from the same incompatibility group cannot replicate within the same cell
▪ It contains practical yet non-essential genes
o Antibiotic resistance
▪ Virulence plasmids → can result in pathogenicity, ex:
o Ti-plasmids → Agrobacterium tumefaciens can induce tumors in plants
o Degrading plasmids → enable bacteria to degrade specific chemicals
▪ Size: 1 – 250kb
▪ Can be a lot of copies from the same plasmid present in one cell = copy number
▪ They can be transferred (ex conjugation)
o Can lead to horizontal transfer (see further) vertical transfer (to descendant)
▪ Biotechnical purpose
o Use modified (recombinant) plasmids as vectors → add new traits to genome of prokaryotes (see 1.6)
Eukaryotic genomes
▪ Chromatin = double stranded DNA spun around histone = DNA-histone complex
o DNA packaging goes trough a set of phases before becoming the chromatin form
1. Nucleosoom = DNA double helix (- chargeis bound to histones (+ charged)
2. ….
3. Chromatine: 2 forms based on how densely packed and therefor how active the genes are
a. Euchromatine → loosely packed → light color → can be transcribed = readable (active)
b. Heterochromatine → densily packed → dark color → inaccessible
4. Chromosome → only visible during the metaphase of cell-divising (X-form)
▪ Chromosome = condensed form of the chromatin = 1 or 2 chromatids → sits in nucleus
o These are linear (but compacted)
o Humans = 46 chromosomes (23 homologues pares) = 46 chromatids
o Chromosomes mostly exist out of 1 chromatid, only just before cell-division you get 2 chromatids, because the
chromatids have been replicated and the two identical copies stay attacked
and thus you get the well-known X form of a chromosome which only exist for a brief period
▪ Diploïd cells = each chromosome is presented in two homologous pairs = having two complete sets of chromosomes
o In most cells of most eukaryotes
o haploid = cells having only one chromosome set
Eukaryotic genome = sum of all chromosomes (nucleus genome + organelle genomes)
It’s size is not related to the complexity of a species = C-value-paradox → 2 reasons
1. Enormous abundance of non-coding genes (humans have capacity for 3 million genes, we only have 30 thousand genes)
a. Recent discoveries: large part of non-coding regions do have important roles
b. Non-coding ≠ non-functional all of the time
2. Presentation of large intron sequences and the occurrence of highly repetitive genomic regions
Organisms with larger genomes contain more intergenic DNA, more introns, more repetitive DNA
Organelle genomes
▪ Animals: mitochondrial DNA = mtDNA | plant: additional plasmid DNA like chloroplast DNA = cpDNA
▪ Organelles are mainly build out of gene products encoded in nuclear DNA, some parts out of their own DNA
, ▪ Mitochondrial and chloroplast genomes are genetic systems = can execute their replication, transcription and protein synthesis
independent from the nucleus. The translation happens in organelle for nuclear DNA, this happens in the cytoplasm
▪ Organelle genomes are mostly circular and come in many copies
▪ Organelle genomes have an exclusive maternal origin because sperm cells have almost no cytoplasm and organelles
▪ Organelle DNA does not undergo genetic recombination
▪ All of this makes organelle DNA useful as molecular markers for specific genetic analyses (see 4.2)
Genome structure of eukaryotic genomes (here for the human genome)
30% Related to genes
▪ DNA connected to protein-coding genes, they regulate how genes behave
▪ Introns = also in the gene next to exons but they are removed during splicing
▪ Promotor = where RNA polymerase binds to start transcription
▪ Enhancers/silencers = regulate gene expression
1-1,5% Protein-coding genes
▪ Tiny fraction of DNA that code for proteins
▪ Exons = part of the protein-coding gene that stay in the final mRNA (introns gone)
20% Unique genes, not gene-related
▪ It’s not repetitive but has no known function and is not part of any gene
50% Repetitive DNA
▪ DNA that appears many times in the genome, scattered or in long blocks
▪ Tandem repeats = satellite-DNA
• Ribosomal RNA-genes = rRNA-genes
o Genes that code for rRNA → ribosomes
o True genes but they code for rRNA, not proteins
• Non-coding RNA-genes
o Genes that code for ncRNA → tRNA, snRNA, snoRNA, miRNA, lncRNA
o These RNAs perform regulation, translation, splicing, …
o Often expressed at telomeres or centromeres
▪ Transposable elements = selfish DNA
• DNA that mostly exist to copy itself
• Retrotransposons → Alu (a SINE) = 106 copies in human genome, 10% of whole genome
o Special: LINES and SINES
• DNA transposons
Repetitive DNA
Consist of typically 50% of the eukaryotic genome
Tandem repeats = satellites
Certain motive like (AAA or CAAC) is repeated fir a number of times e.g. 10 times CA = (CA)10
Based on length of motive:
o Mini satellite DNA (1 – 10bp motive → 10 – 100bp length) → random place
= Short Tandem Repeat (STR) = Simple Sequence Repeat (SSR)
o Micro satellite DNA (10s motive → 100bp – 10kb length) → mostly sub telomeric
o Macro satellite DNA (100 – 1000 motive → up till 1000 kb length) → mostly centromeric
, Transposable elements (TE) = mobile DNA – elements = jumping DNA
Characterised by: inverted repeat sequences (IR) = DNA/RNA where its sequence is followed by its reserve compliment
e.g. 5’ TTAC …. GTAA 3’ = ⊳ ….. ⊲ except for LINES and SINES
Transposons can lead to mutations when inserted in genes, some are activated in stressed situations leading to enhanced
mutation rates → can be positive cause can cause adaptation to a new environment
→ transposons can be used to intentionnaly create genetic mutants
o DNA-transposons
▪ They can ‘jump’ around in the DNA via 2 mechanisms
• Conservative transposition = cut & paste → excision (cut out by transposase + re-insertion)
→ transposase recognises ⊳ sequences = inverted terminal repeat = ITR-sequence
• Replicative transposition = copy & paste → copy that is inserted elsewhere
o Retro-transposons
▪ They jump via copy & paste mechanism via a RNA-intermediate
• Retrotransposon DNA is transcribed into RNA
• Reverse transcriptase turns it into DNA
• Then it gets inserted into a new region
▪ They encode their own reverse transcriptase
▪ Most retro-transposons contain long terminal repeats = LTR = long sequence which is the same at both
ends in the same orientation (e.g. 5’ TTAC ….. TTAC 3’) but way longer (6 – 7 kbp) ITR
▪ There are two exceptions which have no LTR (Long and Short Interspersed Sequences)
• LINES → e.g. LINE-1 → more than 50 k copies in human genome
• SINES → e.g. Alu → more than 500k times present in human genome
Multi-gene families
Multigene family = set of related genes in the genome with similar sequence and related functions originating from a gene duplication
Higher organisms typically have multiple copies of very similar genes by gene duplication = omnipresent in eukaryotes
copies tend to cluster together
when recently duplicated, they have identical function but after a while they can have different functions
over evolutionary time, the accumulation of mutations can lead to diverging function, expression
or even loss of function called pseudogenes = gene copie that after a lot of mutations becomes non-fucntional
Why do gene functions exist?
o Sometimes a lot of copies are needed of a gene because its product is needed in big quantities
▪ E.g. rDNA cause a lot of rRNA is needed
▪ Histones are also much needed
o Gene duplication allows functional diversification of the family members → globin genes
▪ Cause the different members of the gene family (part due to gene duplication) can evolve trough mutations
and selection leading to different structure and or function
Beautifull example: the globin genes – how duplication can lead to new functions
They code for proteins that bind oxygen (leghaemoglobin, myoglobin, hemoglobin) → ontstaan uit 1 gen vroeger
We have as humans a 𝛼-globin cluster on chromosome 16
One gene was copied multiple times, initially identical, now diversed
o Some mutated and stayed functional → changes in oxygen binding
o Others became pseudogenes or got special functions
,How can new genetic variants be formed?
1. Homologous recombination (cross-overs)
a. The homologous chromosomes (one from father and mother) can recombine in the metaphase
b. After replication the chromosome (one chromatid) gets two chromatids = sister-chromatides (on same chromosome)
c. The recombination is between two non-sister chromatids (one of each chromosome)
2. Non-homologous changes
a. Translocations: movement of large chromosomal fragments
b. Non-homologous end joining: repair after dsDNA break
3. Small nucleotide changes in the genome
a. Mutation (A → C)
b. Deletions (GGCTT → GGTT)
c. Duplication (ATC → ATCATC)
d. Insertion → e.g. a transposon jumps in
Horizontal gene transfer
HGT = horizontal gene transfer = LGT = lateral gene transfer
Organism incorporates DNA from another unrelated organism into its own genome
E.g. conjugation between bacteria (same or inter-species) → antibiotic resistance genes
E.g. transformation = taking up loose DNA
E.g. transduction when virus takes bacterial genome piece and takes it to another one
E.g. transfer of mitochondrial genes to nucleus (in cell itself!)
E.g. plastids and mitochondria in eukaryotes
How is HGT or LGT detected?
o By detecting gene/genes in species/genus that is not detected in related species or genera
o And those gene/genes are very similar to a gene from an unrelated species
o Via whole genome sequencing discovered that eukaryotic genomes can acquire this too
1.2. Gene expression: transcription and translation
In prokaryotes
Gene definition in prokaryotes: gene = ORF
Considered as genes in the genome
Genomic sequence that codes for a single protein via ORF
Genomic sequence that results in non-coding RNA (tRNA, rRNA)
Considered as non-genes in the genome
Promotors
UTRs = 5’ untranslated regions (transcribed but not translated)
Operon strucure → organising genes
Operator → regulates transcription
o DNA sequence between promotor and gene or overlapping promotor
o Binding site for repressors to turn of transcription
Promotor
o DNA-sequence upsteam
o Regulates multiple genes = polycistronic expression (prokaryotes) or one also possible (monocistronic)
o Structure (relative basic)
▪ -35 box
▪ TATA-box (situated at -10 = 10bp upstream of the transcription start site)
o Must be recognised by RNA polymerase (RNAP) to start transcription → goes until terminator
One or more structural genes
, o Controlled as a unit
o Transcribed as a whole → one polycistronic mRNA
o Possibility of regulatory genes
Terminator
Regulatory genes
o They code for RNAs or proteins that control the expression of other genes, mosty in another operon
o Like repressor, activator, transcription factor
Regulation of operator: sits between the promotor and genes and will regulate the transcription due to transciptionfactors
→ Repressor; induces transcription: there are two possible mechanisms
1. A repressor protein sits on the operator and switches it off the transcription → OFF
a. An inducer (ligand) binds to the repressor and takes it off → ON
b. Example: lac operon in E. coli where repressor binds operator by default, inducer = allolactose
2. A repressor protein sits on the operator but needs a co-repressor (ligand) to switch it off → ON
a. A co-repressor also binds to the operator switching the transcription off → OFF
b. Example: Tryptophan operon in E. coli where repressor needs tryptophan as co-repressor to switch it off
→ Activator; induces transcription: activates an operator by binding it (more repressor = negative regulation in prokaryotes)
Polycistronic mRNA is one mRNA molecule that was created by transcribing multiple genes
Each gene on the mRNA can be translated independently into a separate protein (e.g. in E.coli → codes for 5 different enzymes)
Each gene has its own
o Shine-Dalgarno sequence → ribosome binding site RBS upstream of start codon
o Start codon (AUG) → translation begins
o Stop codon (UAG, UGA, UAA)
o Open reading frame = ORF = from start – stop codon in codon steps
→ codes for amino acid sequence of a protein
Prokaryotic mRNA does not undergo splicing and processing cause they don’t have introns
In eukaryotes
Gene definition in eukaryotes: not just gene = ORF cause they contain exons and introns (and therefor also undergo processing and
splicing) so its hard to find all ORF in sequenced genomes + there are also non-coding genes (no ORF, and thranscribed into non-coding
RNA) so new definition: gene = part of the DNA that encodes an RNA molecule (mRNA, tRNA, rRNA, other non-coding RNAs)
For eukaryotes a gene = one transcriptional unit with a dedicated promotor region
→ a promotor can regulate only one gene = monocistronic expression
Transciptional unit = DNA transcribed into one RNA so right for eukaryotes
(can be multiple RNAs for prokaryotes)
An eukaryotic gene has monocystronic expression (one promotor for one gene) but I can
lead to one or more gene products
Via alternative splicing of the mRNA for example → one gene leads to several slightly different proteins
Via a gene producing a polyprotein that can be cleavaged in multiple smaller proteins or peptides
,Transcription is mainly positively regulated in eukaryotes (activator) prokaryotes (repressor)
Which means that in normal circumstances, a gene is inactive (OFF), explanations:
(like repressor, sometimes activator is always bound but needs a co-activator to fully switch on the transcription)
Inactivity of a eukaryotic gene is a consequence of the DNA being wrapped into chromatin
o Most promotors are inaccessible for polymerases
o Activation of chromatin (euchromatin) is needed for gene expression
Eukaryotes have a lot of genes so keeping them all on would require a lot of energy
Eukaryotes are mostly multicellular and not every gene needs to be active in every cell
Promotor of eukaryotes
Considered as part of the gene prokaryotes
Most of the time complex: promotor sequence + several enhancer regions or cis-acting/regulatory elements where
transcription factors can bind → they are mostly upstream but can be downstream as well
Processing
Activation promotor → transcription whole gene → pre-mRNA → processing → mRNA → can be alternative splicing (cleaving
the mRNA) → leaves to cytoplasma → ribosome → protein → can be spliced again
From pre-mRNA → mRNA
o Attaching 5’-cap → translation in ribosome starts here cause it binds here
o Introns are removed, only exons stay in
o Attaching 3’ polyAtail
Possible alternative splicing
Translation
Ribosome binds to the 5’-cap
Ribosomes scan towards the Kozak sequence (which includes the start codon) → translation starts
!!Start codons also codes for an AZ
Ends with the stop codon → doesn’t code for and AZ
Protein localization
Protein localisation = expressed proteins accumulate at a give site to go to their defined compartments or cellular regions
Made possible by presence of specific AZ sequences within the protein structure.
E.g. many membrane-bound proteins have signal peptides which are recognised bu signal receptors that guide them
E.g. the nuclear localization site
1.3. Transcriptome
Transcriptome = set of all RNA transcripts (coding as non-coding) in an individual, tissue or cel
embraces all RNA-molecules in cell or population, coding as non-coding
60% of human genome is transcribed into RNA, only 1,5% encodes a protein, the others function as RNA (non-coding RNA), so many
transcripts are not translated, there function is largely unknown
RNA is very hard to study in laboratory cause its unstable, heat-sensitive and easily degraded
We use a trick in labo’s by making a DNA copy of the RNA via Reverse Transcriptase (RT)
This DNA copy of the RNA is called cDNA
Advantages of cDNA over working with chromosomal DNA
o cDNA does not contain introns (cause pre-mRNA → processing → mRNA → cDNA via RT)
But only contain the UTR and protein-coding regions
o cDNA only represents those genes that are being actively used by the cell (transcribed into mRNA)
We can use cDNA therefor to study of a gene is expressed in a cell + quantity of cDNA gives the level of expression
, Making of cDNA (also see practical courses)
1) Extracting all of the mRNA (mature) from the cell = RNA-extraction
2) Then we need to add primers cause Reverse Transcriptase
needs a primer on the mRNA
a. Euk: an oligo(dT)-primer that is hybridized on the poly-A-tail
b. Pro: random hexamer primers are supplied as starting point for RT
3) Also dNTPs need to be added as bui lding blocks for the DNA caused by RT
4) RT then makes a DNA strand complementary to the mRNA strand (U → T)
(see also section 3.3 and section 6.4.)
cDNA library contains all the all genes expressed by a cell at a certain time
1.4. Levels of gene regulation
Gene expression is mainly regulated at the level of transcription initiation (can RNA polymerase start synthesizing mRNA or not)
But additional processes also tightly control every step within the transcription and translation process
1) Regulation of transcription
a. Control of initiation → most important
b. Attenuation → prematurely stops transcription
2) Post-transcriptional regulation
a. Affecting stability/integrity of the mRNA
i. Via microRNAs (miRNAs) or Small Interfering RNAs (siRNAs)
b. Changing the end product
i. Via alternative splicing
3) Regulation of translation
a. Ribosome binding site (Pro: shine-Dalgarno sequence, Euk: Kozak sequence) can effect mRNA translation
b. RNA-binding molecules (like miRNAs) can inhibit the translation
4) Post-translational regulation
a. By adding certain chemical group, the stability and integrity of proteins can be altered
→ glycosylation, methylation, phosphorylation, …. (see also cell biology)
Gene silencing
= a gene present in the genome but which isn’t expressed and therefor not translated into a protein
Transcriptional gene silencing (TGS)
o Gene isn’t expressed cause of the lack of inactivity of the promotor which could be cause by the fact that it isn’t
accessible for transcription factors (activator, repressor, …) due to its presents in heterochromatin
Post-transcriptional gene silencing (PTGS) = RNA interference mechanism = RNAi (https://youtu.be/cK-OGB1_ELE) Zie ppt 3 p42↓
o The gene is transcribed but the mRNA will be destroyed of slowed down = RNA interference = RNAi = PTGS
o This RNA interference is induced by dsRNA (RNA!!)
Knock-out
▪ Long dsRNAs (can come from virus, transposons, …) are cut by enzyme Dicer (endonuclease out of the RNase
= turning off a gene
III family) into small dsRNA’s called duplex siRNAs (||)
completely by changing the
sequence: at DNA level
▪ Duplex siRNA binds to a nuclease complex = RISC = RNA-induced silencing complex
(e.g. random mutagenesis, o RISC cleaves the two strands so you become siRNA that then targets its perfect homologous transcript (mRNA) by
CRISPR-Cas) base pairing with it
o Then the mRNA is cleaved 12 nucleotides from the 3’ terminus of the siRNA
Knock-down
o This mechanism is an important mechanism in their defence against viruses
= turning down a gene at
▪ Release dsRNA → dicer → RISC activated → viral RNA will be degraded → cell survives infection
expression level: at protein or
RNA level
▪ Particularly in low organisms and plants
= gene silencing higher organisms have developed an efficient immune defence (antibodies, killer cells, interferons)
,Gene regulation and its implications for recombinant DNA
The basic concept of recognition of start and stop codon is the same for pro/eukaryotes translation
but transcriptional and translational regulation is fundamentally different in both
This needs to be taken in account when prokaryotes genes are attempted to be synthesized in eukaryotic cell systems and vica
versa
E.g. human insulin gene trying to express in E. coli
o Introns need to be removed (could be by taking mRNA of the gene and turning it into cDNA)
o Prokaryotic elements need to be added (Shine Dalgarno, terminator sequence, …)
Blessing: codon → AZ is universally conserved (so you get the same insulin with the same code in pro/euk)
1.5. Basic techniques for DNA-analysis
Gel electrophoresis
DNA (molecules) are loaded on a gel an can be separated according to their size (nucleotide length)
DNA is attracted to the positive pole
The bigger the molecule, the slower they move trough the gel matrix
bigger molecules have a higher negative charge so in water all the DNA molecules would move at the same speed but
that’s why we use a gel so only the size and not the charge plays a role
DNA ladders are also loaded on the gel (mix of fragments of known sizes) for size estimation
o Loaded in the first (and last) slot of the gel
o Used as size reference
Different types of gels used for electrophoresis which differ in resolving power (to separate) and preparation difficulty
1) Agarose gel electrophoresis
▪ Agarose = polysaccharide from seaweed
▪ The higher the concentration, the stiffer the gel
▪ Easy to prepare + not toxic
▪ Large range of separation → they can separate DNA fragments over a large size-range (200bp – 200kbp)
▪ Low resolving power → they can’t separate a very small difference in size → result in same band
▪ DNA need to be stained for visualisation
o Usually with ethidium bromide or also SYBR, they insert in between the base pairs of DNA/RNA
o Indirect colouring cause they bind with DNA/RNA and are fluorescent under UV
2) Polyacrylamide gel electrophoresis = PAGE
▪ Hard to prepare cause the polymerisation happens under
anaerobic conditions and oxygen inhibits this polymerisation
o Must be poured between glass plates
▪ Acrylamide is a potent neurotoxic
▪ Low range of separation → 50 – 500bp
▪ High resolving power → up to 1 base
▪ Used for separating DNA but more used for separation of proteins
3) Pulsed Field Gel Electrophoresis = PFGE
▪ Used to separate very large pieces of DNA like entire chromosomes → separate more than 1Mbp
So colouring of the DNA/RNA is done by adding ethidium bromide which can then be measured by exposing it to UV-light
It can also be done by adding radioactive atoms (e.g. 32P labelling) which can then be recorded of the gel by autoradiogram
Smears = overlapping bands on the gel in one lane because of incomplete separation of the components
, Restriction enzymes (see also 6.1)
Restriction enzymes = endonucleases type II that can recognise a specific dsDNA sequence = restriction site, within larger DNA molecules
and will cut it within or close to the restriction site, into two smaller dsDNA molecules = restriction framents
A restriction site is named after its 5’ → 3’ orientation so a restriction enzyme which can cut ATTC, can only cut in dsDNA
harboring the sequence 5’ ATTC 3’ → restriction enzyme recognises both
3’ TAAG 5’
Restriction site characteristics
o Often 4,6 or 8 nucleotides long
o Often are these sequences palindromic = complementary (ATTC en TAAG) and reversed (5’ ATTC en 5’ GAAT)
Restriction fragments
o Based on how the restriction enzyme cuts the dsDNA in both strands, you’ll get sticky or blunt ends
o Also different restriction enzymes can have the same recognition site but cleave different → other fragments
DNA sequencing based on method of Sanger
DNA sequencing = determining the nucleotide order of DNA fragment
→ dideoxy method or sanger method
DNA fragment multiplied via PCR
DNA polymerase I → will open dsDNA for replication
1 primer: starting point of replication
Deoxy nucleotides = dNTP for synthesis
Dedeoxy nucleotides = ddNTP with a tag that fluoresces different colours
o They have no 3’-OH
o When reaction starts and a ddNTP is built in, the chain elongation stops
that’s why it’s also called the chain termination method
Then separated all the fragments on a very high-resolution gel electrophoresis → one nucleotide separation
Then scan with laser beam → each ddNTP fluorescens with a different colour → sequence
Usually both strands of the original DNA fragment will be sequenced (once using forward and once using reverse primer)
Problems
Only one specific-sequence can be determined sequenced at a time
You need a primer implying that you need to know the sequence close to the sequence you want to unravel
→ next and next-next generation sequencing avoid these barriers (see 4: high throughput sequencing)
Sanger still fast and cheap and used the most commonly for sequencing (e.g. single PCR product, part of a plasmid, …)
1.6. Basic principles recombinant DNA
Gene cloning
Cloning = creating an identical copy of something
Frequently used to amplify DNA fragments which can be obtained via PCR by using gene-specific primers or restriction
Useful to test this separate gene or produce a large-scale protein production, cause it normally mixed in the cell
Gene cloning = creating copies of recombinant DNA fragment in vivo (in a living organism/cell)
So it’s in vivo cloning (replication) in a host cell of recombinant DNA
Recombinant DNA = DNA molecule created by laboratory methods to bring together genetic material from multiple sources
→ so you create a sequence that can not be found in the genome normally
o Making artificial new combinations of different or parts of genes or DNA fragments
→ restriction on both fragments and combining the sticky ends with DNA-ligase (classic way) (blunt harder)
o Eukaryotic genes can be expressed in bacteria and visa versa if the correct expression signals are present
o E.g. production of human insulin in E. coli
Given by Prof. Tina Kyndt | Els Van Damme | Marjan De Mey
Chapter I
Introduction to the basics (1)
1. Introduction
1.1. Genome
Genome = complete set of genetic material in a cell/organism
Can be analysed in it’s whole by whom-genome sequencing
Functional genome analyses needed to unravel the role of genes within those genomes
Transcriptome analyses to study the expression of certain genes or gene sets
o Transcriptome = embraces all RNA-molecules in cell or population, coding as non-coding
Proteom analyses to find out which proteins are synthesized
o Proteom = full collection of proteins that come to expression in a cel/tissue or specific circumstanses
Interactome analyses to discover how all these molecular interactions are linked
o Interactome = total of macromolecular interactions inside a cel or organism, mainly protein-protein and nucleic
acids-protein interactions
Library = DNA/RNA fragments that you physically store (-80°C) like that or first cloned in E. coli and then stores
Genomic library = contains DNA fragments of an entire genome of an organism → start whole genome sequencing or genome projects
Prokaryotic genomes
The bacterial “chromosome”
▪ Dubble stranded DNA in one single circular structure
▪ Lies in the nucleoid region = chromosome is compacted there
▪ Physical appearance is controlled by supercoiling ad loopforming
o So it becomes accessible for transcription and replication
o So its fits in cells (1 by 2 µm f.e.)
▪ Example E. coli
o 4600 kbp
o 4300 genes → 93% coding genes
o 7% non coding genes → no function or other functions like in the replication (ori for example) and DNA folding
▪ Ori = Origin of Replication = specific DNA sequence where the process of DNA replication begins
On a bacterial chromosome → oriC
o Master switch for the cell’s reproduction, it ensures the chromosome is copied exactly
once before the cell divides
o Usually just one oriC per bacterial chromosome
On a plasmid → oriV or ori
o On plasmids, the ori defines the plasmid’s personality → it determines how many copies
of it can exist (copy number) and which other plasmids can live with it (incompatibility)
o The gene order of E. coli resembles the gene order found in evolutionary related bacteria like Salmonella spp.
▪ Similar gene order on chromosomes of different species = synteny
E. coli and Bacillus subtills that are evolutionary more distinct
,Plasmids
▪ None to some present (copies or compatible different plasmids) in bacteria next to the larger genomic chromosome
▪ Possess their own ori → can replicate independently from chromosome
▪ Sometimes integrated in the chromosome = episome → plasmid replicates together with chromosome
▪ Plasmids can be grouped in incompatibility groups
o Plasmids from the same incompatibility group cannot replicate within the same cell
▪ It contains practical yet non-essential genes
o Antibiotic resistance
▪ Virulence plasmids → can result in pathogenicity, ex:
o Ti-plasmids → Agrobacterium tumefaciens can induce tumors in plants
o Degrading plasmids → enable bacteria to degrade specific chemicals
▪ Size: 1 – 250kb
▪ Can be a lot of copies from the same plasmid present in one cell = copy number
▪ They can be transferred (ex conjugation)
o Can lead to horizontal transfer (see further) vertical transfer (to descendant)
▪ Biotechnical purpose
o Use modified (recombinant) plasmids as vectors → add new traits to genome of prokaryotes (see 1.6)
Eukaryotic genomes
▪ Chromatin = double stranded DNA spun around histone = DNA-histone complex
o DNA packaging goes trough a set of phases before becoming the chromatin form
1. Nucleosoom = DNA double helix (- chargeis bound to histones (+ charged)
2. ….
3. Chromatine: 2 forms based on how densely packed and therefor how active the genes are
a. Euchromatine → loosely packed → light color → can be transcribed = readable (active)
b. Heterochromatine → densily packed → dark color → inaccessible
4. Chromosome → only visible during the metaphase of cell-divising (X-form)
▪ Chromosome = condensed form of the chromatin = 1 or 2 chromatids → sits in nucleus
o These are linear (but compacted)
o Humans = 46 chromosomes (23 homologues pares) = 46 chromatids
o Chromosomes mostly exist out of 1 chromatid, only just before cell-division you get 2 chromatids, because the
chromatids have been replicated and the two identical copies stay attacked
and thus you get the well-known X form of a chromosome which only exist for a brief period
▪ Diploïd cells = each chromosome is presented in two homologous pairs = having two complete sets of chromosomes
o In most cells of most eukaryotes
o haploid = cells having only one chromosome set
Eukaryotic genome = sum of all chromosomes (nucleus genome + organelle genomes)
It’s size is not related to the complexity of a species = C-value-paradox → 2 reasons
1. Enormous abundance of non-coding genes (humans have capacity for 3 million genes, we only have 30 thousand genes)
a. Recent discoveries: large part of non-coding regions do have important roles
b. Non-coding ≠ non-functional all of the time
2. Presentation of large intron sequences and the occurrence of highly repetitive genomic regions
Organisms with larger genomes contain more intergenic DNA, more introns, more repetitive DNA
Organelle genomes
▪ Animals: mitochondrial DNA = mtDNA | plant: additional plasmid DNA like chloroplast DNA = cpDNA
▪ Organelles are mainly build out of gene products encoded in nuclear DNA, some parts out of their own DNA
, ▪ Mitochondrial and chloroplast genomes are genetic systems = can execute their replication, transcription and protein synthesis
independent from the nucleus. The translation happens in organelle for nuclear DNA, this happens in the cytoplasm
▪ Organelle genomes are mostly circular and come in many copies
▪ Organelle genomes have an exclusive maternal origin because sperm cells have almost no cytoplasm and organelles
▪ Organelle DNA does not undergo genetic recombination
▪ All of this makes organelle DNA useful as molecular markers for specific genetic analyses (see 4.2)
Genome structure of eukaryotic genomes (here for the human genome)
30% Related to genes
▪ DNA connected to protein-coding genes, they regulate how genes behave
▪ Introns = also in the gene next to exons but they are removed during splicing
▪ Promotor = where RNA polymerase binds to start transcription
▪ Enhancers/silencers = regulate gene expression
1-1,5% Protein-coding genes
▪ Tiny fraction of DNA that code for proteins
▪ Exons = part of the protein-coding gene that stay in the final mRNA (introns gone)
20% Unique genes, not gene-related
▪ It’s not repetitive but has no known function and is not part of any gene
50% Repetitive DNA
▪ DNA that appears many times in the genome, scattered or in long blocks
▪ Tandem repeats = satellite-DNA
• Ribosomal RNA-genes = rRNA-genes
o Genes that code for rRNA → ribosomes
o True genes but they code for rRNA, not proteins
• Non-coding RNA-genes
o Genes that code for ncRNA → tRNA, snRNA, snoRNA, miRNA, lncRNA
o These RNAs perform regulation, translation, splicing, …
o Often expressed at telomeres or centromeres
▪ Transposable elements = selfish DNA
• DNA that mostly exist to copy itself
• Retrotransposons → Alu (a SINE) = 106 copies in human genome, 10% of whole genome
o Special: LINES and SINES
• DNA transposons
Repetitive DNA
Consist of typically 50% of the eukaryotic genome
Tandem repeats = satellites
Certain motive like (AAA or CAAC) is repeated fir a number of times e.g. 10 times CA = (CA)10
Based on length of motive:
o Mini satellite DNA (1 – 10bp motive → 10 – 100bp length) → random place
= Short Tandem Repeat (STR) = Simple Sequence Repeat (SSR)
o Micro satellite DNA (10s motive → 100bp – 10kb length) → mostly sub telomeric
o Macro satellite DNA (100 – 1000 motive → up till 1000 kb length) → mostly centromeric
, Transposable elements (TE) = mobile DNA – elements = jumping DNA
Characterised by: inverted repeat sequences (IR) = DNA/RNA where its sequence is followed by its reserve compliment
e.g. 5’ TTAC …. GTAA 3’ = ⊳ ….. ⊲ except for LINES and SINES
Transposons can lead to mutations when inserted in genes, some are activated in stressed situations leading to enhanced
mutation rates → can be positive cause can cause adaptation to a new environment
→ transposons can be used to intentionnaly create genetic mutants
o DNA-transposons
▪ They can ‘jump’ around in the DNA via 2 mechanisms
• Conservative transposition = cut & paste → excision (cut out by transposase + re-insertion)
→ transposase recognises ⊳ sequences = inverted terminal repeat = ITR-sequence
• Replicative transposition = copy & paste → copy that is inserted elsewhere
o Retro-transposons
▪ They jump via copy & paste mechanism via a RNA-intermediate
• Retrotransposon DNA is transcribed into RNA
• Reverse transcriptase turns it into DNA
• Then it gets inserted into a new region
▪ They encode their own reverse transcriptase
▪ Most retro-transposons contain long terminal repeats = LTR = long sequence which is the same at both
ends in the same orientation (e.g. 5’ TTAC ….. TTAC 3’) but way longer (6 – 7 kbp) ITR
▪ There are two exceptions which have no LTR (Long and Short Interspersed Sequences)
• LINES → e.g. LINE-1 → more than 50 k copies in human genome
• SINES → e.g. Alu → more than 500k times present in human genome
Multi-gene families
Multigene family = set of related genes in the genome with similar sequence and related functions originating from a gene duplication
Higher organisms typically have multiple copies of very similar genes by gene duplication = omnipresent in eukaryotes
copies tend to cluster together
when recently duplicated, they have identical function but after a while they can have different functions
over evolutionary time, the accumulation of mutations can lead to diverging function, expression
or even loss of function called pseudogenes = gene copie that after a lot of mutations becomes non-fucntional
Why do gene functions exist?
o Sometimes a lot of copies are needed of a gene because its product is needed in big quantities
▪ E.g. rDNA cause a lot of rRNA is needed
▪ Histones are also much needed
o Gene duplication allows functional diversification of the family members → globin genes
▪ Cause the different members of the gene family (part due to gene duplication) can evolve trough mutations
and selection leading to different structure and or function
Beautifull example: the globin genes – how duplication can lead to new functions
They code for proteins that bind oxygen (leghaemoglobin, myoglobin, hemoglobin) → ontstaan uit 1 gen vroeger
We have as humans a 𝛼-globin cluster on chromosome 16
One gene was copied multiple times, initially identical, now diversed
o Some mutated and stayed functional → changes in oxygen binding
o Others became pseudogenes or got special functions
,How can new genetic variants be formed?
1. Homologous recombination (cross-overs)
a. The homologous chromosomes (one from father and mother) can recombine in the metaphase
b. After replication the chromosome (one chromatid) gets two chromatids = sister-chromatides (on same chromosome)
c. The recombination is between two non-sister chromatids (one of each chromosome)
2. Non-homologous changes
a. Translocations: movement of large chromosomal fragments
b. Non-homologous end joining: repair after dsDNA break
3. Small nucleotide changes in the genome
a. Mutation (A → C)
b. Deletions (GGCTT → GGTT)
c. Duplication (ATC → ATCATC)
d. Insertion → e.g. a transposon jumps in
Horizontal gene transfer
HGT = horizontal gene transfer = LGT = lateral gene transfer
Organism incorporates DNA from another unrelated organism into its own genome
E.g. conjugation between bacteria (same or inter-species) → antibiotic resistance genes
E.g. transformation = taking up loose DNA
E.g. transduction when virus takes bacterial genome piece and takes it to another one
E.g. transfer of mitochondrial genes to nucleus (in cell itself!)
E.g. plastids and mitochondria in eukaryotes
How is HGT or LGT detected?
o By detecting gene/genes in species/genus that is not detected in related species or genera
o And those gene/genes are very similar to a gene from an unrelated species
o Via whole genome sequencing discovered that eukaryotic genomes can acquire this too
1.2. Gene expression: transcription and translation
In prokaryotes
Gene definition in prokaryotes: gene = ORF
Considered as genes in the genome
Genomic sequence that codes for a single protein via ORF
Genomic sequence that results in non-coding RNA (tRNA, rRNA)
Considered as non-genes in the genome
Promotors
UTRs = 5’ untranslated regions (transcribed but not translated)
Operon strucure → organising genes
Operator → regulates transcription
o DNA sequence between promotor and gene or overlapping promotor
o Binding site for repressors to turn of transcription
Promotor
o DNA-sequence upsteam
o Regulates multiple genes = polycistronic expression (prokaryotes) or one also possible (monocistronic)
o Structure (relative basic)
▪ -35 box
▪ TATA-box (situated at -10 = 10bp upstream of the transcription start site)
o Must be recognised by RNA polymerase (RNAP) to start transcription → goes until terminator
One or more structural genes
, o Controlled as a unit
o Transcribed as a whole → one polycistronic mRNA
o Possibility of regulatory genes
Terminator
Regulatory genes
o They code for RNAs or proteins that control the expression of other genes, mosty in another operon
o Like repressor, activator, transcription factor
Regulation of operator: sits between the promotor and genes and will regulate the transcription due to transciptionfactors
→ Repressor; induces transcription: there are two possible mechanisms
1. A repressor protein sits on the operator and switches it off the transcription → OFF
a. An inducer (ligand) binds to the repressor and takes it off → ON
b. Example: lac operon in E. coli where repressor binds operator by default, inducer = allolactose
2. A repressor protein sits on the operator but needs a co-repressor (ligand) to switch it off → ON
a. A co-repressor also binds to the operator switching the transcription off → OFF
b. Example: Tryptophan operon in E. coli where repressor needs tryptophan as co-repressor to switch it off
→ Activator; induces transcription: activates an operator by binding it (more repressor = negative regulation in prokaryotes)
Polycistronic mRNA is one mRNA molecule that was created by transcribing multiple genes
Each gene on the mRNA can be translated independently into a separate protein (e.g. in E.coli → codes for 5 different enzymes)
Each gene has its own
o Shine-Dalgarno sequence → ribosome binding site RBS upstream of start codon
o Start codon (AUG) → translation begins
o Stop codon (UAG, UGA, UAA)
o Open reading frame = ORF = from start – stop codon in codon steps
→ codes for amino acid sequence of a protein
Prokaryotic mRNA does not undergo splicing and processing cause they don’t have introns
In eukaryotes
Gene definition in eukaryotes: not just gene = ORF cause they contain exons and introns (and therefor also undergo processing and
splicing) so its hard to find all ORF in sequenced genomes + there are also non-coding genes (no ORF, and thranscribed into non-coding
RNA) so new definition: gene = part of the DNA that encodes an RNA molecule (mRNA, tRNA, rRNA, other non-coding RNAs)
For eukaryotes a gene = one transcriptional unit with a dedicated promotor region
→ a promotor can regulate only one gene = monocistronic expression
Transciptional unit = DNA transcribed into one RNA so right for eukaryotes
(can be multiple RNAs for prokaryotes)
An eukaryotic gene has monocystronic expression (one promotor for one gene) but I can
lead to one or more gene products
Via alternative splicing of the mRNA for example → one gene leads to several slightly different proteins
Via a gene producing a polyprotein that can be cleavaged in multiple smaller proteins or peptides
,Transcription is mainly positively regulated in eukaryotes (activator) prokaryotes (repressor)
Which means that in normal circumstances, a gene is inactive (OFF), explanations:
(like repressor, sometimes activator is always bound but needs a co-activator to fully switch on the transcription)
Inactivity of a eukaryotic gene is a consequence of the DNA being wrapped into chromatin
o Most promotors are inaccessible for polymerases
o Activation of chromatin (euchromatin) is needed for gene expression
Eukaryotes have a lot of genes so keeping them all on would require a lot of energy
Eukaryotes are mostly multicellular and not every gene needs to be active in every cell
Promotor of eukaryotes
Considered as part of the gene prokaryotes
Most of the time complex: promotor sequence + several enhancer regions or cis-acting/regulatory elements where
transcription factors can bind → they are mostly upstream but can be downstream as well
Processing
Activation promotor → transcription whole gene → pre-mRNA → processing → mRNA → can be alternative splicing (cleaving
the mRNA) → leaves to cytoplasma → ribosome → protein → can be spliced again
From pre-mRNA → mRNA
o Attaching 5’-cap → translation in ribosome starts here cause it binds here
o Introns are removed, only exons stay in
o Attaching 3’ polyAtail
Possible alternative splicing
Translation
Ribosome binds to the 5’-cap
Ribosomes scan towards the Kozak sequence (which includes the start codon) → translation starts
!!Start codons also codes for an AZ
Ends with the stop codon → doesn’t code for and AZ
Protein localization
Protein localisation = expressed proteins accumulate at a give site to go to their defined compartments or cellular regions
Made possible by presence of specific AZ sequences within the protein structure.
E.g. many membrane-bound proteins have signal peptides which are recognised bu signal receptors that guide them
E.g. the nuclear localization site
1.3. Transcriptome
Transcriptome = set of all RNA transcripts (coding as non-coding) in an individual, tissue or cel
embraces all RNA-molecules in cell or population, coding as non-coding
60% of human genome is transcribed into RNA, only 1,5% encodes a protein, the others function as RNA (non-coding RNA), so many
transcripts are not translated, there function is largely unknown
RNA is very hard to study in laboratory cause its unstable, heat-sensitive and easily degraded
We use a trick in labo’s by making a DNA copy of the RNA via Reverse Transcriptase (RT)
This DNA copy of the RNA is called cDNA
Advantages of cDNA over working with chromosomal DNA
o cDNA does not contain introns (cause pre-mRNA → processing → mRNA → cDNA via RT)
But only contain the UTR and protein-coding regions
o cDNA only represents those genes that are being actively used by the cell (transcribed into mRNA)
We can use cDNA therefor to study of a gene is expressed in a cell + quantity of cDNA gives the level of expression
, Making of cDNA (also see practical courses)
1) Extracting all of the mRNA (mature) from the cell = RNA-extraction
2) Then we need to add primers cause Reverse Transcriptase
needs a primer on the mRNA
a. Euk: an oligo(dT)-primer that is hybridized on the poly-A-tail
b. Pro: random hexamer primers are supplied as starting point for RT
3) Also dNTPs need to be added as bui lding blocks for the DNA caused by RT
4) RT then makes a DNA strand complementary to the mRNA strand (U → T)
(see also section 3.3 and section 6.4.)
cDNA library contains all the all genes expressed by a cell at a certain time
1.4. Levels of gene regulation
Gene expression is mainly regulated at the level of transcription initiation (can RNA polymerase start synthesizing mRNA or not)
But additional processes also tightly control every step within the transcription and translation process
1) Regulation of transcription
a. Control of initiation → most important
b. Attenuation → prematurely stops transcription
2) Post-transcriptional regulation
a. Affecting stability/integrity of the mRNA
i. Via microRNAs (miRNAs) or Small Interfering RNAs (siRNAs)
b. Changing the end product
i. Via alternative splicing
3) Regulation of translation
a. Ribosome binding site (Pro: shine-Dalgarno sequence, Euk: Kozak sequence) can effect mRNA translation
b. RNA-binding molecules (like miRNAs) can inhibit the translation
4) Post-translational regulation
a. By adding certain chemical group, the stability and integrity of proteins can be altered
→ glycosylation, methylation, phosphorylation, …. (see also cell biology)
Gene silencing
= a gene present in the genome but which isn’t expressed and therefor not translated into a protein
Transcriptional gene silencing (TGS)
o Gene isn’t expressed cause of the lack of inactivity of the promotor which could be cause by the fact that it isn’t
accessible for transcription factors (activator, repressor, …) due to its presents in heterochromatin
Post-transcriptional gene silencing (PTGS) = RNA interference mechanism = RNAi (https://youtu.be/cK-OGB1_ELE) Zie ppt 3 p42↓
o The gene is transcribed but the mRNA will be destroyed of slowed down = RNA interference = RNAi = PTGS
o This RNA interference is induced by dsRNA (RNA!!)
Knock-out
▪ Long dsRNAs (can come from virus, transposons, …) are cut by enzyme Dicer (endonuclease out of the RNase
= turning off a gene
III family) into small dsRNA’s called duplex siRNAs (||)
completely by changing the
sequence: at DNA level
▪ Duplex siRNA binds to a nuclease complex = RISC = RNA-induced silencing complex
(e.g. random mutagenesis, o RISC cleaves the two strands so you become siRNA that then targets its perfect homologous transcript (mRNA) by
CRISPR-Cas) base pairing with it
o Then the mRNA is cleaved 12 nucleotides from the 3’ terminus of the siRNA
Knock-down
o This mechanism is an important mechanism in their defence against viruses
= turning down a gene at
▪ Release dsRNA → dicer → RISC activated → viral RNA will be degraded → cell survives infection
expression level: at protein or
RNA level
▪ Particularly in low organisms and plants
= gene silencing higher organisms have developed an efficient immune defence (antibodies, killer cells, interferons)
,Gene regulation and its implications for recombinant DNA
The basic concept of recognition of start and stop codon is the same for pro/eukaryotes translation
but transcriptional and translational regulation is fundamentally different in both
This needs to be taken in account when prokaryotes genes are attempted to be synthesized in eukaryotic cell systems and vica
versa
E.g. human insulin gene trying to express in E. coli
o Introns need to be removed (could be by taking mRNA of the gene and turning it into cDNA)
o Prokaryotic elements need to be added (Shine Dalgarno, terminator sequence, …)
Blessing: codon → AZ is universally conserved (so you get the same insulin with the same code in pro/euk)
1.5. Basic techniques for DNA-analysis
Gel electrophoresis
DNA (molecules) are loaded on a gel an can be separated according to their size (nucleotide length)
DNA is attracted to the positive pole
The bigger the molecule, the slower they move trough the gel matrix
bigger molecules have a higher negative charge so in water all the DNA molecules would move at the same speed but
that’s why we use a gel so only the size and not the charge plays a role
DNA ladders are also loaded on the gel (mix of fragments of known sizes) for size estimation
o Loaded in the first (and last) slot of the gel
o Used as size reference
Different types of gels used for electrophoresis which differ in resolving power (to separate) and preparation difficulty
1) Agarose gel electrophoresis
▪ Agarose = polysaccharide from seaweed
▪ The higher the concentration, the stiffer the gel
▪ Easy to prepare + not toxic
▪ Large range of separation → they can separate DNA fragments over a large size-range (200bp – 200kbp)
▪ Low resolving power → they can’t separate a very small difference in size → result in same band
▪ DNA need to be stained for visualisation
o Usually with ethidium bromide or also SYBR, they insert in between the base pairs of DNA/RNA
o Indirect colouring cause they bind with DNA/RNA and are fluorescent under UV
2) Polyacrylamide gel electrophoresis = PAGE
▪ Hard to prepare cause the polymerisation happens under
anaerobic conditions and oxygen inhibits this polymerisation
o Must be poured between glass plates
▪ Acrylamide is a potent neurotoxic
▪ Low range of separation → 50 – 500bp
▪ High resolving power → up to 1 base
▪ Used for separating DNA but more used for separation of proteins
3) Pulsed Field Gel Electrophoresis = PFGE
▪ Used to separate very large pieces of DNA like entire chromosomes → separate more than 1Mbp
So colouring of the DNA/RNA is done by adding ethidium bromide which can then be measured by exposing it to UV-light
It can also be done by adding radioactive atoms (e.g. 32P labelling) which can then be recorded of the gel by autoradiogram
Smears = overlapping bands on the gel in one lane because of incomplete separation of the components
, Restriction enzymes (see also 6.1)
Restriction enzymes = endonucleases type II that can recognise a specific dsDNA sequence = restriction site, within larger DNA molecules
and will cut it within or close to the restriction site, into two smaller dsDNA molecules = restriction framents
A restriction site is named after its 5’ → 3’ orientation so a restriction enzyme which can cut ATTC, can only cut in dsDNA
harboring the sequence 5’ ATTC 3’ → restriction enzyme recognises both
3’ TAAG 5’
Restriction site characteristics
o Often 4,6 or 8 nucleotides long
o Often are these sequences palindromic = complementary (ATTC en TAAG) and reversed (5’ ATTC en 5’ GAAT)
Restriction fragments
o Based on how the restriction enzyme cuts the dsDNA in both strands, you’ll get sticky or blunt ends
o Also different restriction enzymes can have the same recognition site but cleave different → other fragments
DNA sequencing based on method of Sanger
DNA sequencing = determining the nucleotide order of DNA fragment
→ dideoxy method or sanger method
DNA fragment multiplied via PCR
DNA polymerase I → will open dsDNA for replication
1 primer: starting point of replication
Deoxy nucleotides = dNTP for synthesis
Dedeoxy nucleotides = ddNTP with a tag that fluoresces different colours
o They have no 3’-OH
o When reaction starts and a ddNTP is built in, the chain elongation stops
that’s why it’s also called the chain termination method
Then separated all the fragments on a very high-resolution gel electrophoresis → one nucleotide separation
Then scan with laser beam → each ddNTP fluorescens with a different colour → sequence
Usually both strands of the original DNA fragment will be sequenced (once using forward and once using reverse primer)
Problems
Only one specific-sequence can be determined sequenced at a time
You need a primer implying that you need to know the sequence close to the sequence you want to unravel
→ next and next-next generation sequencing avoid these barriers (see 4: high throughput sequencing)
Sanger still fast and cheap and used the most commonly for sequencing (e.g. single PCR product, part of a plasmid, …)
1.6. Basic principles recombinant DNA
Gene cloning
Cloning = creating an identical copy of something
Frequently used to amplify DNA fragments which can be obtained via PCR by using gene-specific primers or restriction
Useful to test this separate gene or produce a large-scale protein production, cause it normally mixed in the cell
Gene cloning = creating copies of recombinant DNA fragment in vivo (in a living organism/cell)
So it’s in vivo cloning (replication) in a host cell of recombinant DNA
Recombinant DNA = DNA molecule created by laboratory methods to bring together genetic material from multiple sources
→ so you create a sequence that can not be found in the genome normally
o Making artificial new combinations of different or parts of genes or DNA fragments
→ restriction on both fragments and combining the sticky ends with DNA-ligase (classic way) (blunt harder)
o Eukaryotic genes can be expressed in bacteria and visa versa if the correct expression signals are present
o E.g. production of human insulin in E. coli
