Molecular Genetics Technologies Flashcards
Why is molecular cloning of DNA important?
Purify and amplify fragments of DNA of genes of interest
Obtain DNA sequences
Determine gene structure and regulation
Site-directed mutagenesis to investigate function
Express and purify protein for biochemical/structural analysis
Reintroduce genes into another organism - transgenesis
How was the first artificial recombinant DNA made?
SV40 a lytic virus, wanted to amplify purifies virus DNA
Cut sites but no compatible ends so added polyA and polyT tails using terminal transferase
Filled in gap using DNA pol I
What do these enzymes do? Type II restriction endonuclease Type II methylase DNA polymerase Reverse transcriptase DNA ligase Terminal transferase Polynucleotide kinase Alkaline phosphatase
Cleave DNA at specific sites Methylate DNA at specific points Copy DNA from 3' primer Makes a DNA copy of RNA from 3' primer Joins 2 DNA molecules/fragments Adds homopolymer tails to end of DNA Add a phosphate to 3' end of DNA Remove terminal phosphates from DNA ends
How do type II restriction endonucleases work?
Recognise short DNA sequences and cut at a specific position
Most commonly bind palindromic sequences
Binds as a homodimer forming 2-fold symmetric enzyme-DNA complex
Type II S recognise sequence and cut outside it
How do endonucleases cut DNA ends?
3’ recessed ends
blunt ends
5’ recessed ends
5’ termini have phosphoryl group and 3’ termini have hydroxyl group
What does methylating DNA do?
Restriction enzyme cleavage is blocked by methylating DNA, even if hemi-methylated
DpnI only cleaves methylated DNA
In E. coli Dam & Dcm methylation may result in resistance to cleavage if methylation overlaps recognition site
Some enzymes will not cleave DNA with eukaryotic methylation at CpG dinucleotides
How do DNA ligases work?
Forms 5’ - 3’ phosphodiester bonds in dsDNA
Function to repair strand nicks & join Okasaki DNA fragments
Require ATP
Efficiently joins sticky ends and can join blunt ends
How does the concentration of DNA in ligations effect the outcome?
High concentration gives inter-molecular ligation to produce linear concatenated DNA
Low concentration results in intramolecular ligation to produce circular DNA
Can also link 2 restriction fragments with compatible ends to form a circular piece of DNA - inter then intramolecular ligation
How do DNA polymerases work?
Synthesis DNA 5’ - 3’
They are processive
Many polymerases have 3’ - 5’ endonuclease activity to eliminate errors - DNA pol I has 5’ - 3’ exonuclease
How is Klenow DNA pol I used?
Used to make radioactive DNA probe from entire DNA fragment using random hexanucleotide primers and alpha-32P-dNTP
How does DNA polynucleotide kinase work?
Transfer & exchange phosphate from gamma position of ATP to 5’ hydroxyl terminus of ds or ss DNA & RNA
Used to label 5’ end of restriction fragments
What cloning vectors are available to use in E. coli?
Plasmid derived - vector introduced by transfection
Phage derived - vector DNA introduced by transduction
Combination - vetor DNA introduced by transduction
What are common properties of cloning vectors?
Promote autonomous replication & amplify from single copy Genetic marker(s) to select for/ID cells Unique restriction sites to facilitate cloning of insert DNA Minimal non-essential DNA to maximise size range of cloning
What is the process of molecular cloning?
Insert DNA fragment into cloning site in a vector to produce recombinant DNA molecule - typically by DNA ligation
Introduce recombinant vector into host cell - needs to be circular for E. coli
Recombinant DNA replicated & amplified in cell, expresses antibiotic resistance and can grow in antibiotic
Recombinant DNA passed on after each cell division to produce bacterial colony - clone containing identical copies of introduced recombinant vector DNA
How do you do transformation of E. coli?
Using CaCl2 treated E. coli cells and heat shock
Using high voltage electroporation
How can phosphatase be used to help vectors?
Removes 5’ phosphate
Vector & insert can still ligate together - ligase forms phosphodiester bond in 1 strand and other joined in vivo by E. coli repair system
How are modern plasmids designed?
Modern plasmid vectors (pBluescript, pUC) engineered to replicate at high copy no. minimal size with artificial polylinker cloning region & capability for rapid screening of recombinants
alpha genes have multiple cloning sites for DNA insert
pBluescript - normal cells are blue and cells with insert are white
How does PCR work?
Thermostable DNA polymerase used
Amplifies target DNA sequences defined by specific hybridisation of oligonucleotide primers and their extension by DNA polymerase
Product of reaction used as a template for next cycle - chain reaction
Amplified population of predominantly identical dsDNA
How are nucleotides added to the ends of sequences by PCR?
Added to the end of oligonucleotide primers
These will be added to DNA fragment amplified
How is recombinant DNA constructed using type II restriction enzymes and ligase?
Large number Type II restriction enzymes available to cut DNA to different sizes
Different E. coli vectors available to accept a range of sizes of DNA inserts with convenient multiple cloning site sequences with unique
restriction sites
DNA ends can be modified to enable the joining of incompatible DNA termini
DNA joining position determined by availability of suitable restriction sites - flexibility through PCR
How can you combine DNA molecules by cutting with restriction enzymes?
Ends can be compatible
Fill in/resect DNA ends with T4 DNA pol & dNTPs & perform blunt ligation
Add by blunt end ligation duplex lines containing desired site & cut with restriction enzyme to create cohesive end, or add by blunt end ligation adaptors with preformed cohesive end
Add desired restriction sites by PCR using oligonucleotide primers with restriction sites in 5’ end & then cut with restriction enzymes to create cohesive ends
What is a lambda bacteriophage?
Bacterial virus that lyses and lysogenises if integrated into chromosome
Has viral DNA, 50kb, has sticky cohesive ends of 12bps, linear in phage head - circularises inside cell, cos site is where sticky ends come together in circle
What is the lambda bacteriophage lytic mechanism?
DNA replicates at cos site
Uses ‘rolling circle’ replication
Produces a concatenate cut at cos site
Uses gene A endonuclease to cleave
How is DNA packaged into phage particles in vitro?
Defective lambda lysogens in E. coli so can’t infect phage
Genomes concatenated by ligation & cut at cos sites by lambda protein Ter
Packaged into phage heads using packaging proteins
Artificially constructed phage heads more efficient than transfection
How is bacteriophage lambda converted to a cloning vector?
Normal genome can’t accommodate large inserts as size constraint of fitting into phage head
Can remove section of DNA into involved in lysogenic
Allows inserts up to 10kbp
How can insert size be maximised in lambda bacteriophage?
Non-essential region can be deleted & substituted with ‘stuffer’ region containing restriction enzyme sites to create replacement vector
Stuffer region ensures genome large enough to be packaged into phage
Stuffer region can be removed by restriction enzyme digestion & ligated to new DNA up to 23kbp
What is a cosmid?
Similar to plasmid
Have a lambda cos site
Ligated with fragments of DNA ~35kbp in length
High conc. ligation produces catenane
Phage particles containing cosmid & DNA insert efficiently infected into cells
On entry cosmid and insert circularises
How do we clone a particular gene from a complex genome?
Construct genome/cDNA library
Screen for desired clone
OR
Use PCR to directly amplify up specific sequences from genomic DNA/cDNA made by reverse transcription of mRNA
How are genomic libraries constructed?
Constructed from cloning overlapping DNA fragments - generated by partial digestion of genomic DNA with restriction enzyme
Multiple segment endpoints within the population of fragments - inserts overlap
Partial digest size needs to be compatible with vector insert size capacity
How do you make a genomic library with lambda bacteriophage?
Lambda DNA central region of EMBL3 vector called shutter fragment replaced by insert produced by partial digestion of genome of interest
Plating high titre of infectious phage particles mixed with E. coli
Mixed with soft agar and plated, 1 plaque represents 1 ligation event
Enough plaques to represent entire genome
Can make a filter lift & hybridise with probe OR pick plaques & PCR
What is chromosome walking?
Several clones are detected by probe and can be aligned
Terminal regions of these clones can be used to ID new probes to screen library to ID further clones with overlapping inserts
Overlapping clones constitute ‘contig’
How do you make a genomic library in a bacterial artificial chromosome (BAC)?
BACs = engineered
derivatives of E.coli F
factors
Insert sizes 250–300 kbp, electroporated into cells
Clones robotically picked into 96 well plates
DNAs prepared and IDing specific clone done by
combinatorial PCR screening using gene specific primers
How do you make a cDNA library?
cDNA prepared by reverse transcription of mRNA
Libraries representative of the RNA population
Methylating cDNA with EcoRI methylase
Ligate EcoRI linkers to ends
Cut with EcoRI producing cDNA with EcoRI cohesive ends
cDNA fragments into lambda insertion vectors
How do you screen cDNA library for specific clones?
By hybridisation: DNA/RNA’ oligonucleotide probes
By PCR
Using antibodies to expressed proteins
Functional cDNA screens in appropriate cell types
How is direct cloning of PCR-amplified DNA achieved?
Need some knowledge of
sequence to design primers – can use a pool of degenerate oligonucleotide primers
Target DNA can be genomic DNA/cDNA
PCR product cloned directly into plasmid vectors
TOPO cloning uses vector with T overhang &
topoisomerase linked to ends; combine with Taq PCR product that has an extra A on each end – very efficient
What are multi component plasmids?
Plasmids vectors with DNA sequences from multiple sources for many applications
Reporter expression vectors with fluorescent markers, used to ID cells
Shuttle vectors with separate replication origins, selection markers and promoters
Why do site-directed mutagenesis of cloned DNA?
Introduce single nucleotide changes, insertions, or deletions using synthesised oligonucleotides
Change AA sequence of protein
Create/remove restriction site
Alter gene regulating regions to ID key sequence
What’s the method of site directed mutagenesis?
Parental strands methylated at DpnI sites
Denature parent strands
Anneal oligonucleotide primers and add dNTPs and Pfu polymerase
DpnI enzyme added and only cuts methylated DNA
Even if only one strand cut the strands are destroyed only leaving both strands mutagenised
What are two DNA assembly strategies?
Golden Gate Assembly - utilises type IIS restriction endonucleases to create unique cohesive ends, no restriction sites remaining
Gibson Assembly - allows DNA molecules to be joined via annealing short end regions of complementarity, no sequence ‘scar’, few constraints on application
How does Golden Gate DNA Assembly work?
Type IIS restriction enzyme eg. BsaI, cleaves away from recognition site at diff positions on each strand, results in staggered ends
Positioning of BsaI recognition sites flanking seqs of interest allows DNAs cut to create compatible ends for ligation
Results in seamless ligation product with no BsaI sites left on starting DNAs
Digestion and ligation can occur simultaneously
How does Gibson Assembly work?
Sequence independent seamless joining of DNA molecules, isothermal reaction
Require to link regions A and B from separate plasmids and insert into vector
PCR primers each contain 3’ region of complementarity to plasmid template and 5’ tail with left or right end sequence of other plasmid
5’ ends of PCR products degraded by T5 exonuclease, leaves 3’ single strand regions
Annealing between single strand regions at 50 C
Polymerase - DNA synthesis from 3’ ends to fill gaps
Polymerase catches up exonuclease & nicks sealed by ligase, covalently closed circular molecule
What was the original DNA sequencing methods?
Sanger method
Maxam Gilbert method
Both depend on fractionation of ssDNA in denaturing polyacrylamide gels to give 1 nucleotide size difference resolution
How does Sanger sequencing work?
1 dNTP labelled
Ratio dNTP & ddNTP in each reaction determines probability of chain termination at each position: uses template strand and incorporates dNTPs (high probability) or ddNTPs (low probability), 1 min for each base and run on gel
How does dye terminator Sanger sequencing work?
Can do all reactions at once in one gel as 4 fluorescent dyes used
Can be run through capillaries, automated, use T7 polymerase
Limit of 500-1000 good sequence
How does clone fingerprinting work?
Use genome molecularly cloned in BAC library, overlapping clones ID’d
Each BAC cut into many smaller random fragments & ‘shotgun’ cloned into plasmid vectors
Cloned DNA in plasmids sequenced using universal primer & final sequence assembled from sequence overlaps
What are the common principles of next generation sequencing?
All rely on methods that sequence DNA directly
Genomic DNA reduced to small random DNA fragments by sonication - represent all sequences of starting DNA
DNA fragment library produced and modified in various ways by adapter ligations and by PCR
Rely on massively parallel sequencing in single procedure
Each reaction occurs on discrete solid phase clonal amplification of single DNA molecule - polony
Simultaneously monitored by detector system
How does Illumina sequencing work?
Sequencing by synthesis w/ DNA polymerase & fluorescent terminators
Short ss oligonucleotides of 2 defined sequence (P5 &
P7) chemically bonded via 5’ with 3’ ends free to glass slides to form a dense lawn
Adapters w/ same sequence as bonded oligonucleotides ligated to each end of sonicated DNA - ligated so all have different adaptor
DNA population denatured & added, attaches via complimentary base pairing to bonded oligonucleotides - primers
DNA pol & dNTPs added & DNA molecule copied from 3’ end by P5 & P7
Original DNA washed away
Copy under renaturation conditions to adjacent P5, next round DNA synthesis
dNTPs added for sequencing w/ diff fluorescent groups & reversible termination block
What are the applications of next generation sequencing (NGS)?
Whole genome re-sequencing Gene expression ChIP sequencing microRNA discovery Targeted re-sequencing
What are 2 methods for studying chromatin sequencing?
DNase I hypersensitivity: map open regions of chromatin structure, open = regulatory regions like enhancers/promoters, open regions cut by DNase I, detected by probe
ChIP: detects sequences associated w/ specific chromatin modifications/binding of specific transcription factors, immunoprecipitated DNA detected by region-specific PCR primers
How can NGS be used to analyse chromatin?
Can generate huge no. sequence reads & can be used as sequence ‘counters’
To understand content of DNA extracted a short read/pair of reads of sequence generated from each original molecule obtained from chromatin sampling
25 – 35 bp sequence allows informatics methods to map location to reference genome
