Genetic Variation Flashcards
1
Q
Why study genetic variation?
A
- to determine genetic basis of inherited diseases or interesting aspects of phenotype
- to study relatedness of individuals
- to study the relationships of populations and the degree of intermixing of populations (population genetics)
- to identify individuals (wildlife ecology)
- criminal forensics
2
Q
RFLP analysis
A
- using restriction enzymes to detect DNA polymorphism
- mutations can either create or destroy restriction enzyme sites
- the gain or loss of restriction sites can be detected using gel electrophoresis
- restriction site polymorphisms are most commonly caused by single nucleotide polymorphisms (SNPs)
- restriction enzyme digests of DNA and gel electrophoresis can also be used to detect length polymorphisms (indels)
3
Q
Restriction endonuclease digestion of simple vs complex DNA molecules
A
- a typical RE with a 6-base recognition sequence might cut a 10kb plasmid a few times
- resulting DNA fragment pattern on an agarose gel could be easily determined using a DNA stain
- the same RE would cut genomic DNA several thousand times, producing fragments of varying sizes
- when genomic DNA digest is subjected to gel electrophoresis, and visualized with a DNA stain, the thousands of DNA fragments of varying size produces a smear
- one way to resolve small numbers of DNA fragments within the smear is to use a southern blot
4
Q
Southern blot
A
- invented by Ed southern 1970s
- genomic DNA cut with 1 or more REs
- resulting DNA fragments are size-fraction on an agarose gel, producing a smear
- gel with DNA is treated with an alkali solution to denature the DNA
- DNA fragments are transferred to a membrane (nitrocellulose or nylon) by a blotting process that relies on capillary action to move DNA from gel onto membrane
- the membrane is dried, causing the DDNA fragments to irreversibly bind to it
- the membrane is immersed in a hybridization buffer containing single stranded DNA or RNA fragments (the probe) that are complementary to DNA sequences of interest in the gel
- the probe molecules are labeled, either with radioactive 32P or a chemical label that produces a light signal
- the membrane is exposed to the labeled probe molecules for several hours
- then the unbound prob is washed away, leaving only the probe that has selectively bound to complementary sequences bound to the membrane
- the probe DNA target complexes are detected by autoradiography or chemical methods
5
Q
Applications of southern blots
A
- detecting or localizing genes or other DNA sequences of interest within complex genomic DNA
- eg. Using a probe of a gene from one species to detect the same gene in DNA of another species
- relies on fact that DNA sequence of homologous genes will be quite similar in different species, especially if closely related
- could also detect multiple copies of same gene within a species
- can also detect genetic variation within species due to restriction site polymorphisms or indels (known as RFLP analysis)
-indel variation is the basis of DNA fingerprinting
6
Q
DNA fingerprinting
A
- invented by Alec Jeffreys in 1985
- probed human genomic DNA using a probe containing a repetitive DNA sequence
- discovered that each individual DNA sample produced a unique profile of bands
- probed a southern blot of human DNA (digested with RE) with radioactively labeled repeate containing DNA cloned from human DNA
- obtained a complex multi band profile
- multi band profile for each individual was different from those of others: DNA fingerprint
7
Q
Molecular genetic base of DNA fingerprints
A
- human genomic DNA contains many repetitive DNA sequences which are often grouped in tandem arrays
- the tandem arrays exist in all individuals, but the number of copies of the repeating units varies between people, and between the homologous tandem array on both maternal and paternal chromosomes
- Jeffreys called the tandem arrays minisatellites
- also known as variable number tandem repeats (VNTRs)
- VNTRs are prone to mutations in which DNA repeats are gained or lost
- inherited in Mendelian fashion
8
Q
DNA fingerprinting now done with STRs
A
- as invented by Jeffreys is effective but has disadvantages
- slow and requires large amounts of DNA for the southern blots
- now replaced by a PCR-based method that uses microsatellites (short tandem repeats)
- microsatellites are tandem arrays of short 1-6b repeats found in eukaryotic genomes
- like minisatellite VNTRs, microsatellites are prone to mutation involving gain or loss of repeat units
9
Q
Microsatellite STR genotyping
A
- PCR primers in flanking sequence
- amplify products of different size
- separate products by electrophoresis
- primers are fluorescently labeled
- genotypes identified by size of products
- co-dominant: heterozygous produce 2 bands (both alleles are detected)
- often semi-automated using same capillary electrophoresis machines used for dideoxy sequencing
- multiple STRs often amplified at same time, using primers labeled in different fluorescent colours. Called multiplex Analysis
- 13 standar STR loci are used in criminal forensics. These detect enough variability to distinguish between all human individuals except identical twins
10
Q
STR DNA fingerprinting in criminal forensics
A
- PCR based microsatellite genotyping requires only tiny amounts of DNA: ideal for criminal forensics
- DNA based methods have exonerated many innocent suspects and helped convict real criminals
- methods are so sisitive that contamination can be a problem
11
Q
A few microsatellite/STR loci cause disease
A
- most have no effect on health; they are selectively neutral
- but a few in humans cause disease
- involve trinucleotide repeats within genes or other important DNA sequences
- all humans have these STR loci; however, healthy humans have versions (alleles) with a small number of repeats
- diseased humans have versions with too many repeats which cause production of abnormal proteins
- eg. Huntington’s, myotyonic dystrophy, fragile x syndrome
12
Q
SNPs
A
- single nucleotide polymorphisms
- most abundant polymorphisms in genomes of humans caused by SNPs
- on average a SNP occurs every 800-1000bp
- any 2 randomly chosen humans will have different SNP alleles at several million SNP loci
- usually do-allelic
- at least 85 million SNPs have been catalogued in human genomes from genome sequencing studies
- SNPs that are close to eachother on a chromosome are usually inherited together forming haplotypes
- a haplotype is an arbitrarily long stretch of DNA characterized by particular alleles at the SNP position in that sequence
- current technologies allow many SNPs to be genotyped simultaneously
13
Q
RFLPs
A
-due to SNPs in restriction sites
14
Q
SNP chips
A
- used to genotype large numbers of SNPs
- SNP chips (microarrays) designed to allow many SNPs to be genotyped at once
- synthetics oligonucleotides corresponding to both alleles of known SNPS are arranged on glass slides in a grid pattern
- genomic DNA fragments are fluorescently labeled and hybridized to the probes on the microarray
- binding pattern of DNA fragments is detected with a micro scanner
15
Q
Uses of SNP chips
A
- some important traits/diseases caused by single gene mutation but some caused by many genes
- if certain SNPs or combinations of SNPs are associated with the occurrence of the trait then they likely lie in chromosomal regions close to important genes that are involved
- this can be used to find the genes involved, predict likelihood of having a trait/disease etc
- sometimes the causative mutation of a particular trait/disorder is known and this mutation can be incorporated as a SNP
