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Genes and Gene Cloning
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- 1C Value enigmaAll free organisms use DNA as genetic material - genome = entirety of hereditary information
Prokaryotes much smaller (<10 million bp), complex eukaryotes larger - humans 6 billion, flowering plants 50x larger than human genome
DNA content doesn't relate to organism complexity fully
Amount of DNA genome constant across individuals of species but varies over species - 2C0t analysisMeasure DNA complexity
Denature DNA with heat, slowly cool to allow it to re-anneal
Rate depends on DNA concentration - use known concentration
Determine rate when half DNA reannealed
Simple (many repetitive) = low C0t= quick annealing
Complex (many different) = high C0t= slow annealing as more time for homologous sequences to find each other
dC/dt = -kC^2
low C0t = simple sequence, high C0t = complex DNA - 3C0t curvesVisualise complexity
3 components: rapidly reassociation for very repetitive; more slowly reassociation for moderately repetitive; fraction representing unique that's very slow
E.coli less complex than mammal, but more than T4 bacteriophage - 4Composition of human genome1.5% protein coding exons
26% introns of protein coding genes
702.5% repetitive DNA including transposable elements, simple sequence repeats, segmental duplications and pseudogenes
Large, much repetitive FNA, organisms with huge genomes have more "junk" DNA, not more coding capacity - 5Cloning GenesDNA cleaved with restriction enzyme and placed in vector cut with same enzyme, DNA ligase joins
Recombinant DNA propagated into E.coli for many copies to be made
Genomic library = collection of unique clones containing set of fragments containing all the genome
Some fragments cannot be carried e..g repetitive DNA unstable in E.coli
For representative library, sufficient clones needed to find DNA needed, depends on genome size and amount of DNA in each vector - 6Different genome librariesBAC vectors = 100kb, 140,000 clones
YAC vectors= 500kb, 28,000 clones
Lambda vectors=20kb, 700,000 clones
Bacterial Plasmids:
E.coli library = 11500 clones needed
Human genome = 6.9x10^6 clones needed - 7Analysing DNARestriction enzyme mapping, gel electrophoresis, Southern blotting and sequencing
- 8Gel electrophoresisDigest DNA with restriction enzymes
DNA markers loaded onto gel
Load DNA samples onto agarose gel
Apply current - negative charge on DNA moves towards anode (+)
Smaller move further through gel - separation based on size using marker fragments - 9Southern BlottingAnalysis of genomic DNA
Fractioned DNA transferred to nylon membrane via capillary action, sucked through gel and trapped in membrane
Can detect specific sequences within smear by interrogating membrane with labelled DNA probe by hybridisation
Can see the position of the probe in the hybrid duplex - 10HybridisationIdentify specific clones in genome library
Colonies or plaques transferred to filter
Plate kept for retrieval for positive clones as not all colony transferred
Filters hybridised with labelled DNA- positive clones identified by autoradiography and recovered from OG plate - 11Polymerase chain reactionRevolutionised how low at DNA- allows amplification
Relies on multiple rounds of DNA synthesis, initiated by primers
At the end of each synthesis, DNA denatured to single strands and temp lowered to allow primers anneal and polymerase make new copy of each strand
Thermostable DNA polymerase (prevent denaturation) from thermophilic bacteria e.g. Taq polymerase from Thermus aquatics
At end of each cycle, DNA doubles, 20 cycles = 1 million copies
N cycles forms 2^N copies (theoretically) - 12DNA sequencingCritical for molecular biology
Sanger developed Dideocy chain termination sequencing
DNA chain extended 1bp at a time when synthesising from specific primer
If ddNTP used, DNA synthesis prevented as no hydroxyl group to form phosphodiester bond
4 reactions set up, etc with known single strand template, small radioactive tracer, and specific primer
Each contains small amount of 1 ddNTP- occasionally incorporated into DNA synthesis and stop it going further
DNA synthesis terminated at every base present (1 in each tube) and all initiated form same primer hence set of molecules differing at single base
resolved on polyacrylamide gel, read sequence if all read - 13Improving the experimentMethods remains same
Fluorescently labelled markers - different colour each base- so pool reactions after DNA synthesis and fractioned on same gel lane- more reliable than radioactive based
Fluorescent ddNTPs enable single tube reaction- accelerating rate of sequencing of genomes - 14Sequencing technologiesNow highly automated
Output automatically interpreted by computer - determines sequence nand assesses quality
More than a genome of data needed to control replication errors
Next Generation Sequencing facilitate increase in sequence acquisition rate - 15Illumina Sequencing TechnologyUses DNA polymerase, but has reversible chain termination chemistry
DNA bound to surface of flow cell
Each molecule amplified in situ - cluster of 1000 identiacal copies
Only fluorescent terminator dNTPs present (dye on OH position)
Only one fluorescent dNTP added to growing strand each cycle (terminates)
Laser excites fluorophores on all fragments and captures image
Repeated to build up sequence, one base at a time for 50-100b
Each flow sequence collected from millions of different template DNA clusters - "massive parallel sequencing" - 16Sequencing todayIllumina can do 100 billion bases per day (in 2005, only this many available in data banks)
In one year, single machine can generate 100x more than over past 30 years
Still issues of assembly as only generates 50-150 bases - 17Sequencing GenomesFirst genome 1976 - small RNA phage MS2
2001 first human genome
Clone based approach
Random shotgun approach - 18Methodical clone based approachLarge insert clones (lambda or BAC)
Overlapping set of clones from genomic library form contiguous representation of genome
Select smallest set of cloned that covers region of genes and further break down BACs and sequence
Know where each bit of DNA from so can stitch together easily, but slow
Detailed clone map and easier for assembling repetitive DNA
Sequence already aligned to genome - 19Whole genome shotgunEntire genome randomly fragmented
Lots of small bits of DNA sequenced without reference to organisation of genome
Computer algorithms compare sequences and assemble
Fast, computationally more problematic, difficult to assemble repetitive dNA- much in eukaryotes
Difficult to align into long contigs - 20Repetitive DNA complicates assemblyIF fragment contains DNA sequence present elsewhere, hard to align
More sequence or scaffolds needed for assembly
Paired ned sequence with fragment of known size
Longer reads - 21Genome coverageSequencing not error free - poor quality near start and end of runs
Corresponds to bits of DNA most useful in assembly
Much more DNA needed for single coverage generated
High quality - every base pair present in at least 10 independent reads - 22Prokaryotic geomesRelatively small - under 10mB
Little repetitive DNA
Computer assembly with shotgun easy
Can assemble with low coverage if high quality sequence of relative available
Pairwise comparison (prokaryotes and eukaryotes) allows identification fo gene loss/ growth/ rapid evolution - 23Eukaryotic GenomeFirst sampled = Caenorhabditis elegans
(nematode)
Then Drosophila melangoster- shotgun assembly assisted by already having extensive clone map in fly genome - 24Breaking larger genomesBAC clone
Label 1 end and screen library
Continue to generate continue with each clone consecutively
or
Restriction enzymes to digest lots of clones and define overlap by automated gel imaging - 25Human Genome~ Key dates1983- construction of chromosome-specific libraries
1990- BAC library construction starts
1992- low res genetic linkage map made
1995- physical chromosome Mao
1997 - Bermuda agreement, all sequence freely available
1999- 1st chromosome done
2001- first draft published
2003-sequencing complete
2006- genome finished - 26Key points99.999% accurate
Humans have 20,418 genes - less than expected (alternative splicing- mammalian complexity through regulation of gene expression and splice forms)
WGS not feasible for de novo human genome without scaffold from public projects
WGS mostly assembled on related scaffold e..g chimps and human - 27Longer readsBeing used for gap closure
Oxford Nanopore Sequencing- ss dNA through protein nanopore
Current across pore changes in a way characteristic of different base
Ultralong continuous reads
Live read out and portable - use in field - 28Next Gen SequencingMAss accumulation of sequence data and capacity to relatively easily determine individual human genomes at low cost
Huge computational infrastructure to store data
Social issues - who has access? - 29Annotating genomesProkaryotic simple- genes not interrupted
Tougher in higher eukaryotes - poor understanding of sequences regulating e.g. differential splicing
Introns can be large hence hard to join genes up
Genes can be nested in other genes e.g. hiding in introns
Can use sequence of mRNA and comparative analysis with less complex genomes
Estimates of gene numbers constantly changing - 30Comparative genomesGEnome sequences compared across organisms
Important in bacteria - can identify sets of genes used in pathogenicity
In mammals, reveals rearrangement of genome if evolution
synthetic blocks = segments of chromosomes carrying same genes- help in annotation and inferring gene function
Analysis of closely related conserved sequence helps determine regulatory sequences
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- (Genetic Revolution) Biology of Cells IA Fichas sobre Genes and Gene Cloning, creado por Alice Hathaway el 18/02/2019.
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- Alice Hathaway
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