L16: Genetic Analysis of Human Disease Flashcards
How is human disease analysed using genes?
Using variations between our genomes to track genetic lesions and susceptibility alleles
What are the consequences of crossing-over in multiple generations?
An ancestor with a chromosome containing a genetic variant with an increased risk of disease can be passed down through many generations which has been mixed due to crossing-over meaning there are individuals with the gene in sequence variations that co-segregate with the variant
What are VNTRs?
Variable Number Tandem Repeats that are in many places throughout our genome which can vary between individuals due to unequal cross-over and replication errors
What are microsatellites?
-Short sequence repeats (SSRs) or short tandem repeat (STRs)
-Short repeating units of ~5 nucleotides or less
-Repeated less than 100x
-Repeated number frequency varies between individuals
-Found throughout genome
-Useful for genotyping
What are minisatellites?
-Repetitive GC-rich, variant repeats of 10-100 bases
-Variants are interspersed
-In humans, 90% are at the sub-telomeric region of chromosomes
How is DNA fingerprinting used now in comparison to the original technique?
Now: PCR used to detect numbers of repeats at various loci and amplify DNA
Originally: Southern blot used, probe corresponding to particular minisatellite sequences
Where are minisatellites used?
In paternity testing, bands show the most closely related individual using mother and childs DNA
What is DNA-17 profiling?
It is used for human identification application, allows co-amplification (multiplex PCR) and four-colour fluorescent detection of seventeen loci (sixteen STRs and Amelogenin (version of genes on X&Y))
What are SNPs?
Single Nucleotide Polymorphisms which are single base differences (used when looking for association across the genome)
What are RFLPs?
Restriction Fragment Length Polymorphisms
How is a gene identified on a DNA sequence?
CpG islands are found:
Regions of DNA highly enriched in CpG sites
Frequently associated with 5’ region of genes
identified when sequence is not known since sites for restriction enzymes cluster in CpG-rich regions
How was the Cystic fibrosis gene found?
Using linkage and RFLP analysis, causative mutations and associated genes were found
What type of analysis allowed the discovery of the cystic fibrosis gene?
Positional cloning
What is the major CF mutation?
A 3 nucleotide deletion which lead to the loss of a single amino acid (phenylalanine 508) from the protein
What is a haplotype?
A set of linked polymorphic markers
How can SNPs be used?
Useful to locate genes associated with phenotypes
Diagnosing potential problems/phenotypes
What is the international hapmap project?
Effort to generate comprehensive SNP maps of the human genome, many SNPs are inherited together in blocks called haplotypes
How is the hapmap used?
Can be used for genotyping individuals (most common haplotypes occur in all human populations)
What are the disadvantages of using the hapmap?
Care has to be taken when comparisons are done to get matched control and case samples as the frequencies can differ among populations
What is the main method for SNPs analysis?
BeadChips/GeneChips - applicable to large scale screening, can test 100’s of thousands of SNPs at a time
What are BeadChips and how do they work?
PCR amplification of whole genome
fragment DNA to smaller pieces and hybridise to specific primers bound to beads on chip
Each primer ends 1 base before the position of a known SNP
Extend the primer by 1 nucleotide - nucleotide added will depend on the base in the hybridised DNA fragment (nucleotides labelled)
Read in scanner - identifies base added
What is the GWAS approach?
Genome-wide association study:
-Collect large number of case and control samples
-Put through assays for SNPs spread across genome
-Analyse data for signification association of particular variants with cases rather than controls
-Once association is found use other SNPs in the region of interest to confirm association
How can information be used from the studies using SNP analysis?
-Informs patient care/screening
-Identification of genes and hence pathways that influence particular phenotypes
-Informs about human history
-Insurance
