Human Genome Flashcards
What are the major classes of human DNA sequences?
Nuclear genome and mitochondrial genome
The nuclear genome is split into - genes (25%) and extrageneic DNA (75%)
Genes: coding DNA and non-coding DNA - pseudogenes, gene fragements and introns
Extragenic: unique/low copy number and moderate/highly repetitive - tandem/clustered repeats and intersepersed repeats
What are some points about the major classes of human DNA sequences?
Only about 2.5% of nuclear DNA (10% of 25%) codes for functional gene products
Note that are only around 21,000 genes in the human genome, not 80,000 as above
The non-coding is archival and likely left from retroviruses
Some can have a function in DNA structure i.e. Centromere, telomere
What is involved in the major extragenic repetitive DNA?
Tandem repeats:
Satellite DNA - simple sequence, alpha/beta and localised in heterochromatin
Minisatellite DNA - telomeric/hypervariable family, it is repeated almost as many times as we have genes and localised to telomeres
Microsatellite DNA - found in all chromosomes (good marker)
Interspersed repeats::
Alu family - found in euchromatin, giemsa-positive bands
Kpn (L1) family - found in euchromatin, giemsa-negative bands
Describe polymorphic DNA sequences?
Polymorphism involves one of two or more variants of a particular DNA sequence
They can vary in sequence composition and size between different people
Present in extragenic sequences
Closely linked to defined markers (gene, PCR primer etc.)
Single nucleotide polymorphisms (SNPs) are polymorphisms at a single nucleotide
What can particular polymorphic DNA be linked to?
Mutated (diseased) genes
Minisatellite sequences (also termed VNTR, variable number of tandem repeats) have been used to determine paternity (A Jeffreys, Leicester)
Microsatellite sequences (dinucleotide repeats, also termed short tandem repeats or STRs) also used to map mutant genes and detect individuals
How could we detect VNTRs?
Variable number tandem repeats or Minisatellite sequences
Multi-locus probes (MLPs): detect many different but related sequences
There are many bands in profile so the chance of unrelated people sharing bands is relatively high (1 in 4)
This used to be used in paternity testing
Single locus probes (SLPs): each probe identifies one specific sequence
Use of 3 or more SLPs give low chance of unrelated people sharing band
Used to identify spermatozoa from a vaginal swab to see if it matches a semen swab
How is current DNA profiling carried out?
STR (Short tandem repeat) sequences targeted and PCR is used to amplify the STR sequences
PCR products are analysed by capillary electrophoresis
In the UK a panel of 10 STR sequences are used, estimated to produce spurious match with a probability <10^-13
LCN (low copy number) amplification - amplifies DNA from very small cell numbers (e.g. Finger touch area)
Problem of contamination and ‘alleles drop out’ (failure of PCR to amplify some STRs)
How can we detect inherited diseases at the DNA level?
Indirect detection Linkage analysis (linking polymorphic DNA markers with disease)
Direct Detection
Detection of deletions, insertions (PCR, Southern blotting)
Detection of point mutations - using:
Mutation-specific restriction endonuclease
Allele-specific oligonucleotides (ASO)
Oligonucleotide-linked ligation assay (OLA)
Exploitation of PCR (Amplification-refractory mutation system, ARMS)
Single-strand chain polymorphism (SSCP)
What are some examples of inherited diseases due to single gene defects?
Point mutations
Sickle cell haemoglobin (haemoglobinopathy) - detected by MstII, as it cleaves at CCTNAGG
Alpha1-antitrypsin (emphysema and liver cirrhosis)
Deletions
At least 70% cases of cystic fibrosis - there is a 3bp deletion (Phe) in the CFTR, detected by PCR
Most cases of muscular dystrophy
Certain forms of thalassaemia
Insertions LDL receptor (low density lipoprotein receptor) – certain forms of hypercholesterolaemia
Expansions of a particular repeat
Certain neurological and neuromuscular diseases e.g. Huntington’s Disease is an expansion of a CAG sequence in the coding region of the huntingtin protein
What assays can we do to link genes and disease markers - for known mutations?
By finding allele-specific nucleotides
We need to know the DNA sequence of the mutation being tested for
Hybridisation probes
OLA - oligonucleotide ligation assay
ARMS - Amplification refractory mutation system
Describe the assay using hybridisation probes to find allele-specific nucleotides?
The principles of action - where we hybridise an oligonucleotide sequence to a DNA target
When completely base paired = hybrid is stable
When there is a single mismatch = hybrid is unstable
We have to define the hybridization and washing conditions precisely
E.g. temperature, salt concentration etc
We need to label the oligonucleotide (32P, biotin etc)
Describe OLA?
Oligonucleotide ligation assay
DNA ligase will join the oligonucleotides
The joining will depend on normal probe binding with a common probe = normal OLA product
OR a mutant probe and a common probe = mutant OLA product
The common probe contains a dye label - which can therefore be detected
Describe commercial OLA detection?
This is an analysis performed of multiple OLA products
The size of the product is being detected
Oligos are fluorescently-labelled and products analysed by capillary electrophoresis (like DNA sequencing)
Peaks of fluorescence are detected/produced
Describe ARMS?
Amplification refractory mutation system
We need to know the mutated sequences for PCR primer design
Oligonucleotide primers will be created containing the mutation
The mutation-specific bases are at 3’ end of primer
Gel electrophoresis can then show which mutants are present and which alleles they have
What can be used to test for unknown mutations?
Single-stranded chain polymorphism (SSCP)
This is used to test for unknown mutations
PCR amplify the fragement containing the mutation = millions of copies
Separate the strands of PCR products by gel electrophoresis
The fragments should migrate differently depending on their chain length - as mutations will allow the chains to fold differently
Compare the normal and mutant gels with your own fragement to see which alleles are present
