L16: Genetic Analysis of Human Disease

Question 1

How is human disease analysed using genes?

Answer 1

Using variations between our genomes to track genetic lesions and susceptibility alleles

Question 2

What are the consequences of crossing-over in multiple generations?

Answer 2

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

Question 3

What are VNTRs?

Answer 3

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

Question 4

What are microsatellites?

Answer 4

-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

Question 5

What are minisatellites?

Answer 5

-Repetitive GC-rich, variant repeats of 10-100 bases
-Variants are interspersed
-In humans, 90% are at the sub-telomeric region of chromosomes

Question 6

How is DNA fingerprinting used now in comparison to the original technique?

Answer 6

Now: PCR used to detect numbers of repeats at various loci and amplify DNA
Originally: Southern blot used, probe corresponding to particular minisatellite sequences

Question 7

Where are minisatellites used?

Answer 7

In paternity testing, bands show the most closely related individual using mother and childs DNA

Question 8

What is DNA-17 profiling?

Answer 8

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))

Question 9

What are SNPs?

Answer 9

Single Nucleotide Polymorphisms which are single base differences (used when looking for association across the genome)

Question 10

What are RFLPs?

Answer 10

Restriction Fragment Length Polymorphisms

Question 11

How is a gene identified on a DNA sequence?

Answer 11

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

Question 12

How was the Cystic fibrosis gene found?

Answer 12

Using linkage and RFLP analysis, causative mutations and associated genes were found

Question 13

What type of analysis allowed the discovery of the cystic fibrosis gene?

Answer 13

Positional cloning

Question 14

What is the major CF mutation?

Answer 14

A 3 nucleotide deletion which lead to the loss of a single amino acid (phenylalanine 508) from the protein

Question 15

What is a haplotype?

Answer 15

A set of linked polymorphic markers

Question 16

How can SNPs be used?

Answer 16

Useful to locate genes associated with phenotypes
Diagnosing potential problems/phenotypes

Question 17

What is the international hapmap project?

Answer 17

Effort to generate comprehensive SNP maps of the human genome, many SNPs are inherited together in blocks called haplotypes

Question 18

How is the hapmap used?

Answer 18

Can be used for genotyping individuals (most common haplotypes occur in all human populations)

Question 19

What are the disadvantages of using the hapmap?

Answer 19

Care has to be taken when comparisons are done to get matched control and case samples as the frequencies can differ among populations

Question 20

What is the main method for SNPs analysis?

Answer 20

BeadChips/GeneChips - applicable to large scale screening, can test 100’s of thousands of SNPs at a time

Question 21

What are BeadChips and how do they work?

Answer 21

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

Question 22

What is the GWAS approach?

Answer 22

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

Question 23

How can information be used from the studies using SNP analysis?

Answer 23

-Informs patient care/screening
-Identification of genes and hence pathways that influence particular phenotypes
-Informs about human history
-Insurance