GENOMICS Flashcards
Why use DNA polymerase for enzyme restriction?
PCR
Generate probes
Blunt endings of DNA
Why use polynucleotide kinase for enzyme restrictions?
Used to phosphorylate chemically synthesised DNA - can be ligated to another fragment.
Used to sensitively label DNA so that is can be traced using - radioactively labelled ATP or fluorescently labelled ATP
Features of plasmid vectors
Can be linearised at 1 or more sites in non-essential stretches of DNA
Can have DNA inserted into them
Can be re-circularised without loss of the ability to replicate
Often modified to replicate at high multiplicity within a host cell
Contain selectable markers
Most are relatively small 4-5kb in size
How are bacterial plasmids used as a vector
The vector and the PCR amplicon of the gene must be cut with restriction enzymes to produce compatible ends
Then joined by ligation using DNA ligase
Functionality of Plasmids
Express the recombinant gene in a living organism of choice
add or modify control elements
alter the properties of the gene product
Why are plasmids an essential part of the tool kit?
Discrete circular dsDNA molecules
Are a means by which genetic information is maintained in bacteria
Genetic elements that exist replicate independently of the bacterial chromosomes and therefore extra-chromosomal
Normally exchanged between bacteria within a restricted host range
The reaction for DNA sequencing
Sequencing reaction Strand separation Annealing primer Extension Chain termination
Describe the steps for DNA sequencing, strand separation and annealing primer
DNA is mixed with dideoxy and deoxy nucleotides
Oligonucleotide primer binds to template
polymerase recognises the primer and creates an initiation complex and start elongation.
How does dideoxy chain termination work?
The steps in dideoxy chain termination
- Have a produce template – done by PCR
- Enzymatic sequencing reaction – DNA polymerase make multiple copies of DNA
- Size separation of products by capillary electrophoresis – sorting by size
- Detection of reaction products – sequential detection of the terminating nucleotide to identify the base
- Readout of sequence – reconstructing the sequence
Principles of genetic linkage
Genetic linkage - tendency to alleles at neighbouring loci to segregate together at meiosis
Cross overs are more likely to occur between loci separated by distance than between loci close together on chroomosome
What is linkage analysis?
Method used to map the location of a disease gene in the genome
Genetic markers that are close to our disease gene will be co-inherited together
Ideal genetic markers
Polymorphic Randomly distributed across the genome Fixed location in genome Frequent in population Stable with time Easy to assay
Mechanism for X-inactivation
The Xist gene is transcribed as a long non-coding RNA from the X-inactivation centre and binds all over the X-chromosome
Histone acetylation removed and histone and DNA methylation occur
Inactive X-chromosome is heterochromatic – Barr body
Tsix is derived by transcription in the opposite direction and antagonises Xist RNA to keep 1 X active
What does transcriptomics do
Discovers the biology of your samples
Classify samples
Predict which class a sample belongs to
Data analysis work flow
1) Feature extraction
2) Quality control
3) Normalisation
4) Differential expression analysis
5) Biological analysis
6) Submit data to public repository
Expression analysis
1) Normalisation
2) Hierarchial clustering
3) Gene filtering
4) Statistical tests
5) Generate gene list
6) Biological interpretation
How do SNP microarrays work
Microarrays hybridise with genomic DNA adjacent to SNPs
The SNP is extended by 1 base that is fluorescently labelled and detected using a high definition scanner
Steps in next generation sequencing
1) DNA library construction
2) Cluster generation
3) Sequencing by synthesis
4) Data analysis
Describe the process of DNA library construction
Shearing - DNA chopped into small fragments
Must repair the ends of fragments - Adapters added
Describe the process of cluster generation
Hybridise DNA library fragment to flow cell - is random.
Must amplify fragments. - bridge amplification to make clusters
Describe the process of sequencing by synthesis
Modified 4 bases with chain terminators, different fluorescent colour dye.
Sequence each single nucleotide 1 cycle at a time in a controlled manner.
Single nucleotide incorporation (DNA polymerase)
Flow cell wash
Image the 4 bases
Repeat for full length sequence
How is RNA sequenced in NGS
RNA first converted to cDNA before library construction.
NGS of RNA samples - show which genes are actively expressed.
Number of sequencing reads = measure gene abundance
RNA seq can discover distinct isoforms of genes
Metagenomics
Study of genetic material covered directly from environmental or biological systems/compartments
Microbiome
Characteristic microbial community occupying a reasonably well defined habitat which has distinct physio chemical properties
Microbiota
Ecological community of commensal and pathogenic microorganisms. Includes bacteria, achraea, fungi and viruses
Work flow of 16S PCR amplification
1) sample collection
2) DNA extraction
3) 16S PCR amplification
4) Sequencing
5) Analysis
In 16S PCR amplification which variable region is chosen?
Choice of variable region determines resolution - longer = greater resolution.
Which controls to use in 16S PCR amplification
16SrRNA gene found in all bacteria
Method sensitive to contamination
How to prevent contamination in 16S PCR amplification
Randomise samples
Note batch numbers of reagants
Sequence negative controls
What is whole genome shotgun metagenomics?
Assess taxonomic diversity in sample
Assess composite gene functions in sample
Unbiased, all micro-organisms
Work flow of whole genome shotgun
Sample collection DNA extraction Whole genome sequencing Sequencing Analysis
How to enrich without amplification - Pre-extraction
Differential lysis of mammalian cells
Enrich for intact microbial cells
Potential bias towards gram-positive bacteria
How to enrich without amplification - post-extraction
Enzymatic degradation of methylated nucleotides target mammalian DNA
Bias against AT rich bacterial genomes
Mechanism of RNAi mediated gene silencing
ShRNA procude 50-70 nucleotides. Once transcribed it exits the nucleus. Cleaved by a nuclease = DICER
Cleaved segments bind to RISC and direct cleavage and degradation of complementary mRNA
How to make a mutant mouse
- Targeting vector constructed and introduced into the nucleus of pluripotent ES cells.
- Gene integrates in the cassette. ES selected.
- Positive ES cells grown to blastocytes and implanted into a pseudo pregnant recipient mouse DNA cassette.
- Flanked both upstream and downstream with DNA sequences that match with site introduced - HOMOLOGENE ARMS
Site specific recombinase
Efficiently cause deletions or translocations, inversions in the genomic DNA with high fidelity
Cre-lox system
Site specific recombinase technology.
Cre-recombinase recombines a pair of lox sequences.
Mechanism of deletion in cre-lox system
LoxP sites face the same direction
Cre-recombinase will detect the unidirectional loxP sites and excise the floxed segment of DNA
Mechanism of inversion in cre-lox system
LoxP sites orientated towards each other.
Region floxed will become inverted
Translation blocking MOs
Bound to 5’ UTR of mRNA
Interfere with ribosomal initiation - prevent translation of transcript
Splice inhibiting MOs
Prevent splice directing small ribonucleoprotein complexes from binding to their targets at the borders of introns on a strand of pre-mRNA
What does CRISPR/Cas9 system do
Guide RNA binds to strand of genomic DNA
Cas9 endonucleases bind to non-protospacer portion of gRNA + PAM of DNA.
DSB 3bp upstream of PAM
Mutations can be introduced through NHEJ and HDR
Mitochondrial DNA replication machinery
Polymerase G
Twinkle
Single stranded binding protein (mtSSBP)
TFAM
Steps in strand displacement model of mtDNA replication
- Replication of heavy strand begins at origin of heavy strand
- Replication of light strand begins at origin of light strand
- Replication of both strands completed
- Segregation of daughter molecule
Describe what happens in the initiation of heavy strand replication
Parental heavy strand displaced and coated with mtSSBP
Twinkle helicase unwinds mtDNA
Mitochondrial RNA polymerase synthesise RNA primer using light strand as template.
Polymerase G use RNA primer to replicate DNA at origin of heavy strand
Describe what happens when light strand begins replication
Heavy strand replication passes to origin of light strand.
STEM LOOP structure is formed preventing mtSSBP binding.
Mitochondrial RNA polymerase synthesises RNA primer using heavy strand as template
Polymerase G uses RNA primer to replicate light strand at origin
Genomic imprinting
Is the selective expression of genes related to the parental origin of the gene copy
Process of imprinted genes
Is mediated by Imprinting control regions (ICRs)
1 copy is silenced by DNA methylation catalysed by DNMT3a and histone methylation - leading to inactivation
Imprinting patterns are reset during gamete formation
Hyperchromicity
Increased absorption of light at 260nm on denaturation
Single stranded DNA absorbs UV light better than double stranded DNA
Tm
Point at which 50% of all strands separate
Things that affect Tm
GC content Length Salt concentration pH Mismatches
Complementarity
Perfect mathces have higher Tm
Perform hybridisation at the Tm of perfectly matched duplexes
Mismatches would be unstable at high temperatures
Stringency
Manipulate conditions
Limit hybridisation between imperfectly matched sequences increases specificity
Determined by temperature near Tm or low salt concentration
Microarrays
Ordered assembly of thousands of nucleic acid probes
Probes fixed to a solid surface, sample hybridised to probes
What do restriction endonucleases do
Recognise specific sequence
Cut that sequence
Limit the transfer of nucleic acids from infecting phage’s into bacteria
Migration
Physical movement of people from a different population = new pools of variants being introduced to an existing population = ADMIXTURE
Cross species comparison
Generate evolutionary profile for a gene or gene family
Cross species conservation
Identify important regions of a gene and its protein
Gene duplication
Duplication of a DNA sequence containing a gene
Mechanism = unequal crossing over
Unequal crossing over
Recombination between sequences that are not correct sequence but are very similar
Alpha globin genes
Chromosome 16
3 genes and 3 pseudo genes
Beta globin gene
Chromosome 11
5 gene 1 pseudogene
Globin cluster evolution
Through duplication and divergence
Globin genes evolved through duplication and accumulation of mutations (divergence)
Some are functioning genes, and some are not (pseudogenes)
Divergence of promoters has occurred so bind different transcription factors and allow expression of genes at different stages of development
Pseudogenes
Typically have mutations and are nono-functional
Many of them in genome
After gene duplication, 1 gene can maintain the original function and the other can diverge
Sickle cell disease symptoms
Anaemia – fatigue, restlessness, jaundice
Acute pain episodes – due to O2 deprivation of tissues
Increased frequency of infections – spleen damage
Also, stroke, pulmonary hypertension, gallstones, liver and kidney problems, joint problems, delayed puberty
Genetics of SCD
single base change in beta-globin gene of Hb A = HbS
Is an autosomal recessive genetic disease. Is a codon change
Natural selection and HbS
2 copies of HbS variant has significant negative effects on reproductive ability – SCD
1 copy of HbS variant confers resistance to severe malaria = heterozygote advantage – HbS variant is maintained in the population
Problems with GWAS
identify associations, however contribution to genetic component is low Many common SNP Rare SNP Copy number variation Epigenetic variation
