Molecular Genetics Flashcards
1
Q
What are the two types of molecular alleles?
A
- Single nucleotide polymorphisms (SNPs)
- Micro and mini satellites
2
Q
What is a SNP?
A
- Single nucleotide polymorphism
- The simplest variation in DNA sequence: a one base pair difference
- Detected via genome sequencing and ASO but mainly via restriction enzymes
3
Q
Why are SNPs detected by restriction enzymes?
A
- The single pase pair chance causes a change in a restriction enzyme site so the enzyme will no longer cleave the DNA at this site
- This means that when a piece of DNA with the SNP and without the SNP will be cut a different number of times by the same restriction enzyme
- Called a restriction fragment length polymorphism (RFLP)
4
Q
What is the process by which RFLPs are detected by restriction enzymes?
A
Can be done by 2 methods:
- Restriction enzyme digestion of DNA and Southern Blotting:
- Digestion of DNA using specific restriction enzyme creates a smear of DNA sequences
- The specific RFLP is detected using a labelled probe by Southern Blotting
- The RFLP alleles are co-dominant (heterozygote is recognisable)
- Outdated method - PCR from genomic DNA followed by restriction enzyme digestion:
- PCR amplifies portion of DNA containing RFLP
- This amplification makes the portion of DNA visible
- The amplified region is cut with restriction enzyme and run on agarose gel
- Much more efficient and rapid
5
Q
How are SNPs that do not cause a change in a restriction enzyme site?
A
- Allele specific oligionucleotide hybridisation (ASO):
1. Use a small probe (~20 bp) that is completely complementary to one allele (100% match) - The second allele contains the SNP which means there will be a mismatch between this probe and its sequence
- At low temps the probe will bind allele 1 and allele 2 (SNP variation)
- When temperature is raised the probe will only bind to the allele that is completely complementary and will not bind the SNP allele
6
Q
What are the advantages of disadvantages of SNPs for molecular mapping?
A
Advantages:
- SNPs are very common in the genome (approx. once every 1000 bp)
Limitations:
- Each SNP occurs as only two alleles (A or T, C or G)
- For DNA profiling there is a target to have as much variation as possible
7
Q
What are micro and mini satellites?
A
- These are regions in the genome that have short sequences of DNA that occur in a variable number of tandem repeats
- Micro-satellite: 2-10bp repeating section
- Mini-satellite: 10-100bp repeating section
8
Q
How are micro/mini satellites detected?
A
- They are detected by gel electrophoresis by:
1. Souther hybridisation using the repeat (or adjacent sequence) as a probe
2. PCR, using the sequences on each site of the repeats as primers - Like other molecular alleles micro/mini satellites are co-dominant, so the heterozygote is visible
9
Q
What are the benefits of using satellite markers for mapping?
A
- They occur about once every 10,000 bp in the genome
- There are many alleles (number of repeats)
- This means there is a high level of polymorphism
- Better method than SNPs
10
Q
How are distances between molecular markers mapped?
A
- do not use term in coupling/repulsion (this is a term for dominant and recessive allele positions only)
- The RF is calculate using the number of recombinants/total no of progeny
- This converts to map units
11
Q
Is the pattern of crossing over the same in human male and female meiosis?
A
- No
- There are hot-spots in recombination in male gametes at either end
- Female recombination is more spread out across the chromosome
12
Q
Can molecular markers be used to map human disease?
A
- Yes, if the molecular marker is closely linked to the disease causing locus
- Beneficial as many disease causing mutations are very complex and difficult to map
13
Q
What is DNA fingerprinting?
A
- Based on differentiating between individuals using minisatellites
- The mini-satellites are found in multiple loci
- People have different numbers of repeats at these loci
- The process involves digesting genomic DNA with a restriction enzyme and then performing a Southern Blot
- Using a probe complementary to the repeat to detect all the repeat loci at once
- This produces a unique pattern of bands- a “fingerprint”
14
Q
What are the pros and cons of genetic fingerprinting?
A
Pros:
- First technique available to distinguish individuals based on their DNA
Cons:
- Southern blots require large amounts of DNA
- DNA must be intact (cant use degraded samples)
- Can be difficult to distinguish bands
15
Q
What is DNA profiling?
A
- A newer technique
- Uses microsatellites (STR loci)
- Uses a single locus
- Uses 10-15 unlinked (seperate chromosomes or far apart on same) microsatellite loci that are 2-4 bp in length and highly variable in copy number
- Uses PCR to detect them (primers bind outside or repeat regions)
