WEEK 4 Flashcards
What are SNPs?
- Single Nucleotide Polymorphisms
- SIMPLEST variant of a DNA sequence is one base pair difference between two alleles
What can SNPs change in the restriction enzyme behaviour?
- The ability for it to cut the surrounding sequence
What is a Restriction Fragment Length Polymorphism?
- Method used to detect small differences (SNPs) by using a restriction enzyme to cut sequence and seeing where it cuts or doesn’t cut
How do we detect RFLP?
- Using PCR from genomic DNA followed by restriction enzyme digestion –> Design primers to then carry out the polymorphism chain reaction.
What is a limitation of RFLP?
- Not every SNP you’re interested in will cut a restriction enzyme site!
What are two methods to detect SNPs that do not cause changes in restriction sites?
- Allele specific oligonucleotide hybridisation (you can identify a SNP in ANY part of the genome
- Allele specific PCR
Why are SNPs good for mapping?
- They are VERY COMMON in the genome
How often does the SNP occur?
- Every 1000 bp in the genome –> Human genome = 3 000 000 kbp so about 3 million SNPs
What is a limitation of SNPs?
- Each SNP occurs as ONLY 2 alleles! and the second category of DNA sequence polymorphism has MANY alleles
What are micro and mini satellites?
- Short DNA sequences that occur in a VARIABLE number of tandem repeats in a genome
What does the number of micro and mini satellite repeats vary between?
- Varies between INDIVIDUALS AND between the two chromosomes in individual
What is a micro-satellite (short tandem repeat, STR) defined as?
- 2-10 base pair repeat of a DNA sequence
What is a mini-satellite (Or Variable Number of Tandem Repeats, VNTR) defined as?
- 10-100 base pair repeat of a DNA sequence
How are mini-satellites and micro-satellites detected?
- Old fashion way= Southern Hybridisation using the repeat sequence as a probe
- New cool way= PCR using sequences on each side of the repeat as primers
What are the satellite alleles inheritance?
- Co-dominant
How often do satellite markers occur?
- Once every 10 000 bp so 300 000 satellite loci
What 3 areas are satellite markers used in due to the many alleles they have (number of repeats) and high level of polymorphism?
- DNA profiling and forensic genetics
- High resolution genetic mapping
- Ecological genetics and conservation biology
In mapping with DNA markers what does the ‘phase’ mean?
- Which alleles are together (eg. A1+B1)
What is the haplotype?
- the genotype for CLOSELY linked genes on a SINGLE chromosome
What are the benefits of genetic mapping?
- Can use to tell if a disorder is caused by one gene or by DIFFERENT genes
- Genes whose DNA sequence is not yet known (only its mutant phenotype) can be cloned from their map location
- Assists with identifying disease loci in combination with the whole exome/genome sequencing
- Nearby markers can be used as a tag of a desired gene in plant and animal breeding (marker assisted breeding)
- Closely linked DNA markers are useful in genetic counselling e..g Huntington disease
If a gene locus and a DNA marker locus are r map units apart, what is the probability that she has inherited the gene disease allele if she has inherited a SNP A4? (i.e. Formula)
1- (r/100)
What are three reasons why DNA fingerprinting and profiling are possible?
- genomic DNA sequence is stable (unless mutation)
- All cells in body have same DNA (unless mutation)
- DNA is UNIQUE (huge variation between individuals) –> humans have a lot of genetic diversity
How can DNA be detected from fingerprinting and how does it work?
- With mini satellite loci
- Digestion of genomic DNA with restriction enzyme and perform SOUTHERN BLOT
- Use a probe COMPLEMENTARY to the repeat to detect ALL repeat loci at ONCE
- fingerprint of bands will come up
Which technique is used to detect DNA from mini satellites?
- A southern blot
What does a higher number of loci mean for DNA fingerprinting with mini-satellite loci?
- That the increased probability pattern will be specific (unique)
What are three problems with DNA fingerprinting?
- Southern blots require large amounts of DNA (several micrograms)
- DNA must be INTACT (can’t reliably use degraded samples)
- Can be hard to interpret –> are similar bands the same allele from the same locus?
What is the method of DNA profiling?
- 10-15 UNLINKED microsatellite loci that are 4 base repeats and highly variable in copy number (highly polymorphic)
- Use PCR to detect –> DNA at one locus is AMPLIFIED (primers bind OUTSIDE repeat)
- Alleles defined unambiguously by the repeat number
What is multiplex PCR?
- Many pairs of PCR primers in one mixed reaction - Some primer pairs labelled with different colour dyes
- Automated detection shows PEAKS instead of bands
- AMPLIFY 10-15 different regions into a single PCR reaction
What are the advantages of DNA profiling?
- PCR is very sensitive, so requires very LITTLE starting material
- Can genotype partially degraded DNA samples –> PCR will ONLY amplify INTACT DNA (degraded DNA will not amplify)
- This means old samples can be used without getting false bands
What is a major disavantage of DNA profiling?
- Contaminated DNA can be easily amplified :/
What is the main result of a DNA profile?
- Probability!
What three things are related to the probabiliy being used to determine if the profile is UNIQUE in DNA profiling resutls?
- Unlikely that related people will share every band
- Each locus is INDEPENDENT so probabilities are multiplied
- Even a low number of loci results in huge/tiny probability e.g. Pr of random caucasian having allele A1 is 1/4, allele D3 is 1/5 and G4 is 1/10.
What does interpreting DNA profile results require?
- Large databases/knowledge of population genetics
Can DNA profiling establish innocence?
- YES
In DNA profiling is exclusion of identity/relatedness easy?
- YES
In DNA profiling, is complete proof of identity/relatedness possible?
- NO it is IMPOSSIBLE
If two DNA fingerprints/profiles match, is this proof that they came from the same person?
- NO but you can indicate a PR that they came from the same person (thus other evidence needed)
What are the two potential sources of error from DNA profiling?
- False inclusion
- False exclsuion
What can false inclusion be due to in DNA profiling?
- Relatives sharing the same alleles
- Some alleles being more frequent in specific populations (founder effects)
- Try to use markers that show no difference in allele frequency
What can false exclusion be due to in DNA profiling?
- Technical problems like ‘allele-drop out’
- Problem with very low amounts of DNA
- Contamination or mixed source of DNA
- human error
What are the differences between DNA FINGERPRINTING and DNA PROFILING?
- DNA fingerprinting uses minisatellites (profiling uses micro)
- DNA fingerprinting uses restriction digest followed by southern blotting (profiling uses PCR to detect alleles from a SINGLE locus)
- DNA fingerprinting uses a single probe to detect alleles from SEVERAL loci (profiling combines several INDEPENDENT microsatellite markers)
- DNA fingerprinting can NOT determine which allele is from which locus
- DNA profiling can calculate the Pr of a profile matching by chance
What are the 4 applications of DNA fingerprinting and profiling?
- Clinical (twins mono or dizygotic)
- Forensic (Comparing crime scene samples with suspects, identification of remains, identifying source of illicit drugs)
- Legal (biologcial father, immigrants being related, GMO crop tracking)
- Conservation biology (Determining source of poached wildlife, effects of climate change on plants)
What do mutations help us to understand?
- Biological processes
How can we genetically dissect biologcial processes?
- By finding or creating mutations that effect the process
What is a mutation?
- A change in the genetic material –> occuring in the germ line is inherited
What does deleterious mean?
- Usually causing harm or damage
- e.g. Mutant allele
What is a forward mutation defined as?
- The change from a WT to a Mutant
What is a reverse mutation or REVERSION defined as?
- The change BACK to WT
How can we increase the rate of mutations?
- Via mutagens (certain chemicals)
What are clones defined as in terms of somatic mutant cells?
- Original mutant cell will be progenitor of a population of mutant cells (these are the clones)
What is an individual who has genotypically different somatic regions?
- A genetic Mosaic
Is there a small or large mutant sector if a somatic cell mutation occurs EARLY in development?
- LARGE mutant sector
- Loss of function is more severe
Is there a small or large mutant sector if a somatic cell mutation occurs LATE in development?
- SMALL mutant sector
- Only SMALL portion
What are chromosome mutations?
- Changes in chromosome structure or arrangement
What are genome mutations?
- Changes in chromosome NUMBER (e.g. Down syndrome)
What are single gene mutations?
- Relatively small changes within a particular gene
What is a base substitution?
- A single base change e.g. A–> T in sickle cell anaemia
What is a transition single gene mutation?
- Where a pyrimidine is replaced by a pyrimidine (C/T) OR a purine is replaced by a purine (A/G)
What is a transversion single gene mutation?
- Where a purine is replaced by a pyrimidine (or vice versa) –> pyrimidine replaced by a purine
What are Indels?
- Inertions or deletions
What do Indels invovle?
- Short DNA sequences (same or more than 1 bp deleted or added)
- Causes frameshift if not multiple of 3
- Also can get transposable element insertions
How large is a short tandem repeat (microsatellite)?
- Anywhere between 2-10 base pairs
What are 3 examples of Human Tri-nucleotide (triplet) repeat disorders?
- Huntington’s disease (CAG repeat)
- Myotonic dystrophy (CTG repeat)
- Friedreichs ataxia (GAA repeat)
Does Myotonic dystrophy affect the protein?
- NO
- It affects the AMOUNT of protein being produced (due to methylation)
If a person contains a higher number of repeats will they have a lower or higher age of onset (e.g. Huntingtons or fragile X)?
- They will have a LOWER age of onset
Is anticipation seen in all children inheriting the allele for huntongtons?
- YES
What is anticipation?
- Where a SINGLE gene disorder can become more severe in subsequent generations.
What do beneficial mutations do?
- enhance the survival or reproductive capacity of organism
What do conditional mutations do?
- Affect the phenotype only under DEFINED CONDITIONS
What is a null mutation?
- Where there is total loss of function (e.g. White eyes in drosophila)
What is a hypomorphic mutation?
- Partial loss of function (e.g. apricot eyes in drosophila)
- Common in autosomal recessive disorders
Are null mutations generally recessive?
- YES
- 50% is enough function
What is haploinsufficiency?
- When 50% function is not enough for the full function of the protein–> Phenotype is seen in the heterozygote (null mutation is dominant rather than recessive)
What are the most common forms of loss of function mutations?
- Dominant negative mutations
What are dominant negative mutations?
- Where a NON-FUNCTIONAL protein is made that INHIBITS function of a normal protein in heterozyotes
- It is a dominant mutation
- Can bind to the functional copy and disable it
in which structures are dominant negative mutations common in?
- Structural proteins that form dimers
What 3 things can a gain of function mutation result in?-
- Gene product GAINS a new function (neomorphic)
- Protein is always active (hypermorphic)
- gene has INCREASED levels of expression (due to inappropriate level of expression, less common and generally dominant)
What are CONDITIONAL mutations?
- Mutations that cause the mutation under CERTAIN conditions, but is NORMAL in other conditions
e. g. temperature sensitive in curly winged drosophila
What does it mean when we say conditions (conditional mutations) are permissive?
- That they allow the normal phenotype to occur (e.g. 20 degrees)
What does it mean when we say conditions (conditional mutations) are restrictive?
- They restrict the normal phenotype (e.g. 25 degrees)
What are conditional mutations useful for determining?
- WHEN a gene is active during development
How is the gene information within a gene accessed? (process)
- In the process of gene expression
What are prototrophs?
- Wild type strains that grow on Minimal Media (contains only a CARBON source, inorganic salts, and biotin)
What are auxotrophs?
- Isolated mutants that disrupt the synthesis of essential molecules so they could only grow on complete media
Where do consensus sequences exist in terms of exons and introns?
- At the exon-intron boundary
Which type of RNAs carry out splicing?
- snRNAs
What is the spliceososme made up of?
- snRNAs and proteins
Which dimensions does the expression of genes vary in ?
- time and space
What changes in chromatin have to occur to allow transcription to proceed?
- Chromatin compaction
Do trasncriptionally active regions of chromosomes have loose or tight DNA-protein regions?
- They have looser DNA-protein regions than inactive regions
What controls the compaction level of chromatin?
- Histones (protein bound around DNA)
What does DNA methylation cause?
-Inhibtion of transcription
Are housekeeping genes (always active) methylated or unmethylated?
- unmethylated
Where are CpG islands located?
- Near the promoters of many genes
What are the cis acting elements that influence transcription? (2)
- Promoter
- Regulatory or control elements
What are the two types of transcription factors that regulatory elements bind?
- Transcription activators
- Transcription repressors
What is the function of transcription activators?
- They bind to DNA regulatory elements called enhancers and INCREASE transcription
What is the function of transcription repressors?
- They bind to DNA regulatory elements called SILENCERS and PREVENT transcription
What is combinatorial control?
- The combination of TFs bound to the regulatory elements of a gene at any time which determines the levels of transcription.
What is spatial control in terms of TFs?
- That they are expressed in certain cells
What can a mutation in a PROMOTER region result in ?
- An increase or decrease in transcription
What can a mutation in splice recognition sites result in?
- Pre-mRNA not being spliced correctly
What can a mutation in the 5’ or 3’ UTR result in?
- Alteration in mRNA stability or in ability of mRNA to be translated
What are 4 ways to regulate gene expression after transcription?
- Altering pre-mRNA processing (splicing)
- Altering mRNA stability (changes mRNA levels)
- Altering mRNA localization within a cell
- Regulation of translation
What is the most common mechanism of post translational regulation?
- Alternative splicing
What does alternative splicing do?
- Regulates which exons are in the final RNA transcript
