Basic genetics in relation to DNA Flashcards
Betathalacaemia
Chemical surgery has been performed on human embryos to remove disease
World first
Uses of genetics
Paternity tests, even for dogs
DNA gifts
DNA picture
Costs of sequencing genome
Dropped dramatically, now can sequence in under a day
Head and neck cancer genome sequence
21% have specific genetic mutation that could be targeted
GeneSight
Tests to see if you are going to react well to depressive/ anxiety/ bipolar medications. Will it help you?
23andMe
Saliva based service
Inherited conditions, drug responses, Genetic Risk Factors, traits
e.g. familial dysautonomia
A lot of money for stupid tests, not very effective –> came into trouble, went into ancestry
What is familial dysautonomia?
Affects development and survival of certain nerve cells
Disorder disturbs cells in ANS, which controls involuntary actions
Personalised genetic testing and teeth
Not engaged enough yet
PerioPredict - can test if you have risk factors for periodontal disease
Overproduction of interleukin 1 gives heightened inflammatory response in presence of bacterial plaque
Cons of PerioPredict
Have to have it to get Dental Insurance in US, dangerous
- basis for whether you have 1 or 2 visits to dentist per year
- dangerous: periodontitis not just related to IL-1, including lifestyle choices
Who was DNA fingerprinting invented by
Alec Jeffreys (Leicester) 198
DNA fingerprinting relies on
Variable number of tandem repeats (VNTRs) - 30-40% of non-coding region
DNA fingerprinting gene
Myoglobin gene - segments in introns with no apparent function, vary in size and composition between individuals
- initially called mini-satellites as surround the exon
- repeating nucleotides (15-100) 1kb - 20kb (no inherited from parents)
- large variation of number of these regions between individuals
How does DNA fingerprinting work
Repeat regions usually bounded by specific restriction enzyme sites
Restriction enzymes - DNA-cutting enzymes (from bacteria)
Cut at specific sequences so gap would be created (ECOR1 one of first restriction enzymes identified)
Run DNA out onto gel - restriction digestion
Gel electrophoresis separates fragments left behind by size
PCR
DNA amplification
- measuring transcription of gene
- starting material is DNA rather than mRNA
- reverse transcription: use RNA to make cDNA and then use polymerase chain reaction to make many copies of specific piece of DNA (then you can detect it)
PCR history
1955 with discovery of first DNA polymerase
1985 Kary Mullis developed polymerase reaction in Taq
DNA likes to stick to DNA and RNA, RNA likes to stick to DNA and RNA (annealing after denaturation)
Then elongate at 72 degrees
Called hybridisation
Amplification
Is exponential, wanted gene can make 68 billion copies in 36 cycles
Need enzyme that works at 72 degrees
Amplify sequences
HE4 expression in AL1 cells require continued retinoic acid mediated differentiation
-Ethidium bromide example of gene expression being associated with phenotype of cell
Problems with standard reverse transcription PCR
- poor precision
- lower sensitivity
- slow dynamic range <2 logs
- non-automated
- size-based discrimination only
- results not expressed as numbers
- Ethidium bromide staining is not very quantitative
Real-time PCR
Automated method
Allows detection of PCR amplification during early phases of reaction, kinetics of reaction not end-point
Exponential phase is optimal point for analysing data
Advantages of real-time PCR
-traditional measure at end-point and real-time collects data in exponential growth phase
-> reporter fluorescence directly proportional to no of amplicons generated
-> dynamic range of detection
no post-PCR processing
-detection down to 2-fold change
Next generation sequencing
Massive parallel sequencing - sequence hundreds of millions of DNA molecules simultaneously
-single NGS run can sequence human genome approx. 37x in 27 hours
Important for personalised medicine
Proteomics
Large scale study of proteins, particularly their structures and functions, includes post-translational modifications
-better because not all genes are translated into proteins?
Saliva
New blood test?
-tests health and wellness, detects oral and systemic illnesses early
Non-invasive
Single nucleotide polymorphism (SNP)
DNA sequence variations when single nucleotide sequence is altered
Must occur in a least 1% of pop., 90% of all genetic variation
Occur every 100-300 bases, 2 out of 3 involve C to T
SNP and alleles
Synonymous SNPs have different alleles that encode for same a.a.
Non-synonymous SNPs re SNPs that have different alleles that encode diff amino acids
Effect of SNPs on cell function
Many have no effect on cell function, others predispose to disease, influence response to drugs to environmental insults
VNTR
Variable Number of Tandem Repeats
Number of repeated segments varies between people
Some only found at single locus, some found at many loci
Polymerase chain reaction
30-40 cycles of 3 steps
- denaturation (1 min 94 degrees C)
- annealing (45s 54 degrees C)
- forward and reverse primers - extension (2mins 72 degrees)
- only dNTPs
Optimum point for analysing data in real-time PCR
Exponential phase
If diff amounts of DNA in starting sampe and if measurements taken at plateau the data would not truly represent the starting material
Real-time PCR detects accumulation of amplicon during reaction
Taqman probe reaction
Piece of DNA with 2 fluorescent tags
Taqman probe and primer bind to target DNA, reporter dye released from extending double-stranded DNA
Reporter dye now distant to quencher so will give off signal (fluoresce)
Signal passes to specific software for analysis
Requires specific probe to bind to a specific sequence
SYBR Green Dye
Binds to any double stranded DNA
Burst of light as DNA is denatured and SYBR Green Dye floats free
Hybridisation
Localisation technique for detection of gene product (RNA) in tissues
Another way to investigate cell-type and tissue specific gene expression
Plunc expression (postnatal mouse)
Found by hybridisation
Restricted to epithelial layers of trachea and main bronchial passages
Expressed in regions of nasopharynx
Transfection
‘Transformation’ + ‘infection’
- infection of cells by isolated nucleic acid from virus, resulting in production of complete virus
- process whereby the nucleic acid sequences are intertroduced by either biochemical or physical processes
- stable or transient transfection
Why do we want to transfect cells?
Protein production for clinical or research applications
Change protein expression profile of cell to assay for effects of gene
-overexpression of gene
-RNAi (reduction in expression of gene)
Addition of genetic markers to cell line
Genomics
Recombinant DNA, DNA sequencing methods, and bioinformatics
Used to sequence, assemble, and analyse function and structure of genomes
Transcriptomics
Genome-wide RNA transcript expression levels
Metabolomics
Comprehensive characterisation of small molecular metabolites in biological systems
Overview of metabolic status & global biochemical events assosiated with cellular or biological system
-can depict both steady-state physiological state of cell or organism & also dynamic responses to genetic or environmental factors
Salivomics
Ability to monitor health & wellness, as well as detect oral & systemic illnesses early through noninvasive means
Use saliva to detect and monitor systemic diseases
Human microbiome
Properties of microbiota or normal flora difficult to study as not always possible to ‘grow’ bacteria in lab and then ‘type’.
Metagenome
Biology and medical significance of human microbiome and its collective genes
Genome-wide association studies
Rapid scanning of complete sets of DNA (or genomes) of many people to find genetic variations associated with particular disease e.g. Human Genome Project
