Genetics Flashcards
define molecular pathology
the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids
The genome contains:
all genes, regulatory sequences and other information contained within an organism’s DNA
Subdivisions of the genome: (2)
Large nuclear genome, very small mitochondrial genome
_% of the nuclear genome is highly conserved during evolution - functionally important
5%
Protein coding DNA sequences account for __% of the genome, the rest is non-coding (but still functional and important)
1.1%
DNA can be selectively amplified via
PCR
List four advantages of using PCR
- cheap & fast (alternative to cloning)
- Targeted - can amplify very specific sequences from small amounts
- can selectively detect DNA sequences not normally present in the tissue being tested (e.g viruses)
- can analyse highly degraded DNA samples
DNA can be separated via __________ according to ___ and ____
DNA can be separated via gel electrophoresis according to size and conformation
List the three types of gels used in electrophoresis and when they are used
- Polyacrylamide gels - single stranded DNA molecules less than 500 nucleotides in size
- Agarose gels - more porous gels for 300-200,000 nucleotides
- Pulsed-field gel electrophoresis - for long DNA molecules
DNA samples move from the ____ charge size towards the ____ charge side.
DNA samples move from the negative charge size towards the positive charge side.
Sanger sequencing utilises _____ dyes to colour nucleotides
florescent dyes
Define genomics
The branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes.
list the advantages of Next Generation sequencing (2)
- quicker - parallel sequencing can produce millions of sequences at once
- reduced cost of DNA sequencing by several orders of magnitude
What is the downside of Next Generation sequencing?
big data = need large storage space
Analysis of RNA allows us to: (4)
- count the amount of RNA made as a readout of the activity of a gene (gene expression)
- read the RNA sequence to find mutations
- Show that DNA translocations create a ‘new’ spliced mRNA (that can then go on to make a ‘bad’ protein)
- Test for mutations affecting exon splicing of the mRNA
What is RT-PCR and its function?
What are its advantages? (2)
reverse transcriptase-polymerase chain reaction - analyses individual gene transcripts/gene ‘expression’ of RNA
- highly sensitive
- cost effective
Why don’t we try to analyse RNA as it is? How do we solve this issue?
RNA is not very stable as a molecule - converting it into dna via RT-PCR is more viable
Real time RT-PCR is used to:
assess the gene expression/abundance of individual RNAs in a sample
Real-time RT-PCR has a single step _____ and _____
amplification and detection
The greater the quantity of target DNA (or copy DNA) in the starting material, the ____ a significant increase in fluorescent signal will appear, yielding a ____ Ct
The greater the quantity of target DNA (or copy DNA) in the starting material, the faster a significant increase in fluorescent signal will appear, yielding a lower Ct
Methods that allow selective amplification are: (2)
PCR & Cloning
Methods that allow specific detection are: (3)
- Southern blot
- Microarray
- Libraries
Define hybridisation
The formation of a double-stranded DNA between two single strands with complementary sequences
Describe the principle/process of hybridisation (3)
- denature strand
- bring in labelled probe
- renature into one strand
List the 6 hybridisation methods and whether DNA or RNA can be used with it.
- Dot Blot (DNA)
- Southern Blot (DNA)
- Fluorescence in situ hybridisation (FISH) (DNA + RNA)
- Single Nucleotide Polymorphism (SNP) arrays (DNA)
- Microarray (DNA + RNA)
- Array Comparative Genomic Hybridisation (aCGH - DNA)
How many genes can RT-PCR test at a time?
only one
Expression Microarray is a method of hybridisation that:
enables the simultaneous analysis of the expression profiles of thousands of genes (in RNA)
There are ______ Single Nucleotide Polymorphisms (SNPs) in your genome
10 million
SNPs serve as landmarks in the search for: (3)
- genes associated with disease risk
- drug responses
- complex phenotypes
How do we identify genes associated with disease? How does this work?
GWAS - geneome wide association studies
- searches genome for genetic variants (SNPs)
- looks at thousands of nucleotide variants at the same time
Array Comparitive Genome Hybridisation (aCGH) can be use to: (2)
- scan a complete genome for copy number imbalances
- detect aneuploidy, deletions and duplications
How do we analyse protein? (3) What do these processes involve?
Western Blot (immunoblotting) - detection of polypeptides after size-fractionation of a tissue lysate on a polyacrylamide gel
Immunohistochemistry (IHC) - studies the overall protein expression across a slice of tissue
Immunoassay - antibodies can be used to quantitate the amount of a protein or antigen in a lysate
Immunohistochemistry can reveal _____ and ______ of proteins but not _____.
Immunohistochemistry can reveal amonunt and location of proteins but not protein size.
Western blotting asseses the ____ and ____ of the protein but not its ____.
Western blotting asseses the amount and size of the protein but not its location.
List the 4 methods of DNA assessment and what can be detected:
- Cloning
- PCR
- Hybridisation (arrays)
- Next-Generation Sequencing
Can detect sequence variation (SNPs and mutations) & Changes in amount of DNA (copy number changes)
List the 3 methods of RNA assessment and what can be detected:
- RT-PCR (and real time RT-PCR)
- Next-Generation Sequencing
- Hybridisation (arrays)
Can detect gene expression (RNA abundance)
List the 3 methods of Protein assessment and what can be detected:
- Immunohistochemistry (IHC)
- Western Blotting
- Immunoassay
Can detect protein abundance/expression, size and cellular localisation
What is a mutation?
A change or variation in the base sequence of DNA
If a germline mutation does not seriously impair ________________, it can spread.
If a germline mutation does not seriously impair an individual’s ability to have children, it can spread.
Most disease associated mutations are _______. These mutations are ______ de novo.
inherited, very rarely
Mutations can be…. (4)
positive, neutral, damaging or lethal
note: not a 4 point scale, but actually a sliding scale
List the two sources of new mutations and how they come to be:
- Endogenous - due to spontaneous errors in DNA replication and repair
- Mutagens/Environement - most mutagens act directly via an ability either to damage a particular nuclotide or to become incorporated into the nucleic acid - this causes causes single/double stranded breaks which need to be repaired and lead to increased chance for error
There is _____ mutation(s) for every _____ cigarettes smoked.
1 mutation for every 15 cigarettes smoked.
List the three types of mutations and give an example for each:
- Chromosome disorders - Patau syndrome
- Single gene (monogenic) disorders - cystic fibrosis
- Complex/multifactorial disorders - cancer, heart disease, diabetes
Chromosome disorders are due to:
an excess or deficiency of the genes contained in whole chromosomes or chromosome segments - includes many genes
List the 6 types of chromosome mutations:
- Translocations
- Deletions
- Duplications
- Inversions
- Chromosome loss
- Chromosome duplications
Define syndrome:
a condition in which many cell types can be affected throughout the body
Why do chromosome mutations result in syndromes?
Chromosomes contain many genes which account for many functions - disruption affects many genes at once and thus results in multiple affected phenotypes
Patau syndrome is also known as ______- and is caused by ______
trisomy 13 - additional gene
always caused by error and cannot be inherited.
Turners syndrome is also known as ______ and is a result of ______
45 XO, results from complete or partial X chromosome monosomy in a phenotypic female
Cri du chat syndrome is also known as _______ and is a result of ______
5p monosomy, results fro the loss of a chromosome arm
Single gene disorders are caused by ______
individual mutant genes
Single gene disorders may be _____ or _____
recessive or dominant
Single gene disorders may be contained in the _____ or _____ genomes.
mitochondrial or nuclear
Single gene disorders usually exhibit obvious ______ patterns
pedigree patterns
The two types of substitutions mutations are ____ and ____. List the functions of each and which one is more common.
Transition - substitution of purines with purines (A/G to A/G) or pyrimidines with pyrimidines (C/T to C/T) - most common
Transversion - purine to pyrimidine or pyrimidine to purine - less common
Transversions are theoretically expected to be ___ as common as transitions but _____ is a hot spot. This is often triggered by _______
2x, C>T, UV damage in cancer - easy to take off a methyl group via deamination from C to T
List the three classes of substitutions and their functions:
- Synonymous “silent” mutations
- no change in AA
- frequent
- often occurs in 3rd base “wobble position”
- sometimes remains unrepaired - Non synonymous/nonsense mutation
- replacement of AA with a termination codon
- dramatic reduction in gene function
- leads to premature protein truncation
- safe to assume pathogenic - Non synonymous/Missense mutation
- replacement of AA with a different AA
List the two types of missense mutations:
conservative: replacement amino acid is similar - minimal effect on function
non-conservative: replacement amino acid is dissimilar - more serious effect on function
List the order of molecular pathology (4)
mutation - altered protein - altered function - disease
Frameshift mutations are + result in:
insertion or deletion of a base/bases - result in an entirely different sequence of AAs from the point of insertion/deletion; usually ends in premature truncation of the protein
The effect of the premature protein truncation in frameshift mutations will depend on: (3)
- the stability of the polypeptide product
- the extent of the truncation
- the functional importance of the missing amino acids
Autosomal recessive inheritance - features in pedigree charts: (4)
- appears in siblings of proband but not in parent, offspring or other relatives
- males/females equally affected
- parents of affected child asymptomatic carriers of mutant alleles
- Recurrence risk for each sibling of proband 1:4
Autosomal dominant inheritance - features in pedigree charts: (4)
- phenotype usually appears in every generation - each affected person with an affected parent
- Child of affected parent has 50% chance of inheriting it
- phenotypically normal family members do not transmit the phenotype to their children
- Males/females equally affected
X-linked inheritance - features in pedigree charts: (5)
examples include:
- incidence much higher in males than females
- heterozygous females usually unaffected/mildly affected due to random X inactivation
- affected male will pass on the gene to all of his daughters, who will be carriers
- gene ordinarily never transmitted directly from father to son
- may be transmitted through a series of carrier females
examples include: androgen insensitivity syndrome, muscular dystrophy, fragile X syndrome, haemophilia
X-linked dominant - features in pedigree charts: (3)
examples include:
- affected males with normal mates; no affected sons and no normal daughters
- male/female offspring of female carriers have a 50% risk of inheriting phenotype
- affected females twice as common as affected males but females typically have milder disease
examples include: retinitis pigmentosa, rett syndrome, vitamin D resistant rickets, congenital generalised hypertrichosis
Y-linked dominant - features in pedigree charts: (3)
- affects only males
- affected males always have affected father (unless there is a sporadic mutation)
- all sons of affected man are affected
What is genetic testing? What molecules does it involve?
The analysis of molecules that present a read-out of an inherited gene disorder being present in an individual - molecules include DNA or RNA to look for the mutation itself (direct) or RNA/protein or certain metabolites (biochemical) as a downstream surrogate
When can genetic testing be conducted? (2)
Diagnostic - after individuals have presented with symptoms
Predictive - when individuals are at risk without any current symptoms
What is Genetic Heterogeneity?
Many different pathogenic mutations leading to the same disease OR more than one gene with many mutations leading to one disease outcome.
What is locus heterogeneity?
Different genes (loci) can each have mutations that lead to the same clinical phenotype
What are allelic heterogeneity?
Where one gene can have many different mutations that lead to the same clinical phenotype - e.g cystic fibrosis has over 1000 mutant allels in the CFTR gene which can each lead to the disease
What is Penetrance?
The probability that having a mutant gene = disease phenotype presents - unlikely to have 100% penetrance
Can penetrance be reduced?
Yes - the effect of a mutant allele can be dampened to reduce penetrance by the inheritance of other modifying genes or environmental factors. Can present in a family as “skipping a generation”
What is expressivity?
Variation within the severity of the phenotype of a genetic defect
List the 3 time periods of predictive genetic testing
- Pre implantation
- Pre-natal
- Pre-symptomatic
What is pre-natal genetic testing?
Genetic diagnosis of fetus during pregnancy and before birth
What is pre-implantation genetic diagnosis (PGD)?
Can only be offered if mother is undergoing IVF - embryos screened for specific genetic features prior to selecting the preferable ones for implantation
- performed by removing a single cell from a forming 8-cell (day 3) or a few cells from a blastocyst (day 5) embryo
What is pre-symptomatic genetic testing?
Occurs after birth but before symptoms of disease present.
Prenatal testing in down syndrome - Non-invasive blood testing looks for what features? (3)
AFP - elevated in neural tube defects, reduced in Down Syndrome
Inhibin A & hCG - high in Down Syndrome
Oestriol - low in Down Syndrome
What is not able to be tested for in chorionic villus sampling?
AFP
