Basic genetics in relation to DNA

Question 1

Betathalacaemia

Answer 1

Chemical surgery has been performed on human embryos to remove disease
World first

Question 2

Uses of genetics

Answer 2

Paternity tests, even for dogs
DNA gifts
DNA picture

Question 3

Costs of sequencing genome

Answer 3

Dropped dramatically, now can sequence in under a day

Question 4

Head and neck cancer genome sequence

Answer 4

21% have specific genetic mutation that could be targeted

Question 5

GeneSight

Answer 5

Tests to see if you are going to react well to depressive/ anxiety/ bipolar medications. Will it help you?

Question 6

23andMe

Answer 6

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

Question 7

What is familial dysautonomia?

Answer 7

Affects development and survival of certain nerve cells

Disorder disturbs cells in ANS, which controls involuntary actions

Question 8

Personalised genetic testing and teeth

Answer 8

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

Question 9

Cons of PerioPredict

Answer 9

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

Question 10

Who was DNA fingerprinting invented by

Answer 10

Alec Jeffreys (Leicester) 198

Question 11

DNA fingerprinting relies on

Answer 11

Variable number of tandem repeats (VNTRs) - 30-40% of non-coding region

Question 12

DNA fingerprinting gene

Answer 12

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

Question 13

How does DNA fingerprinting work

Answer 13

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

Question 14

PCR

Answer 14

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)

Question 15

PCR history

Answer 15

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

Question 16

Amplification

Answer 16

Is exponential, wanted gene can make 68 billion copies in 36 cycles
Need enzyme that works at 72 degrees

Question 17

Amplify sequences

Answer 17

HE4 expression in AL1 cells require continued retinoic acid mediated differentiation
-Ethidium bromide example of gene expression being associated with phenotype of cell

Question 18

Problems with standard reverse transcription PCR

Answer 18

• 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

Question 19

Real-time PCR

Answer 19

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

Question 20

Advantages of real-time PCR

Answer 20

-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

Question 21

Next generation sequencing

Answer 21

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

Question 22

Proteomics

Answer 22

Large scale study of proteins, particularly their structures and functions, includes post-translational modifications
-better because not all genes are translated into proteins?

Question 23

Saliva

Answer 23

New blood test?
-tests health and wellness, detects oral and systemic illnesses early
Non-invasive

Question 24

Single nucleotide polymorphism (SNP)

Answer 24

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

Question 25

SNP and alleles

Answer 25

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

Question 26

Effect of SNPs on cell function

Answer 26

Many have no effect on cell function, others predispose to disease, influence response to drugs to environmental insults

Question 27

VNTR

Answer 27

Variable Number of Tandem Repeats Number of repeated segments varies between people Some only found at single locus, some found at many loci

Question 28

Polymerase chain reaction

Answer 28

30-40 cycles of 3 steps 1. denaturation (1 min 94 degrees C) 2. annealing (45s 54 degrees C) - forward and reverse primers 3. extension (2mins 72 degrees) - only dNTPs

Question 29

Optimum point for analysing data in real-time PCR

Answer 29

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

Question 30

Taqman probe reaction

Answer 30

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

Question 31

SYBR Green Dye

Answer 31

Binds to any double stranded DNA | Burst of light as DNA is denatured and SYBR Green Dye floats free

Question 32

Hybridisation

Answer 32

Localisation technique for detection of gene product (RNA) in tissues Another way to investigate cell-type and tissue specific gene expression

Question 33

Plunc expression (postnatal mouse)

Answer 33

Found by hybridisation Restricted to epithelial layers of trachea and main bronchial passages Expressed in regions of nasopharynx

Question 34

Transfection

Answer 34

'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

Question 35

Why do we want to transfect cells?

Answer 35

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

Question 36

Genomics

Answer 36

Recombinant DNA, DNA sequencing methods, and bioinformatics | Used to sequence, assemble, and analyse function and structure of genomes

Question 37

Transcriptomics

Answer 37

Genome-wide RNA transcript expression levels

Question 38

Metabolomics

Answer 38

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

Question 39

Salivomics

Answer 39

Ability to monitor health & wellness, as well as detect oral & systemic illnesses early through noninvasive means Use saliva to detect and monitor systemic diseases

Question 40

Human microbiome

Answer 40

Properties of microbiota or normal flora difficult to study as not always possible to 'grow' bacteria in lab and then 'type'.

Question 41

Metagenome

Answer 41

Biology and medical significance of human microbiome and its collective genes

Question 42

Genome-wide association studies

Answer 42

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