Genetics Final Flashcards
(148 cards)
1
Q
Chromosome variations
A
- Permanent chromosome variations
- Can be passed to offspring
- two types
2
Q
The two types of chromosome variations
A
- Chromosome rearrangements
- Variation in chromosome numbers
3
Q
Chromosome rearrangements
A
Changes in the structure of individual chromosomes
4
Q
Variation in chromosome numbers
A
Changes in the numbers of chromosomes one or more individual chromosomes deleted or added
5
Q
What is caused by chromosome variation
A
Genetic disorders
6
Q
Four types of rearrangements
A
- duplication
- Deletion
- Insertion
- Translocation (swaps to non homologous)
7
Q
Double stranded breaks do…
A
Cause rearrangements
Are caused by radiation
Can be fatal without repairs
8
Q
Deletions
A
Loss of segment
9
Q
Terminal deletion
A
One break at terminal end
10
Q
Internal deletion
A
Rejoining of incorrect ends (2 breaks)
11
Q
Major effects of deletion
A
Loss of information
12
Q
When can deletion loops be detected
A
Meiosis
13
Q
Deletion consequences
A
- loss of DNA sequences
- phenotypic effects depend on size & location
- Pseudo-dominance
- can effect gene dosage
14
Q
Duplications can be…
A
Tandem, multiple alleles, and moved anywhere
15
Q
Why are duplications important in genetics
A
Copies of genes are raw material for new adaptations
16
Q
What percent of human genomes consists of duplications
A
8%
17
Q
How do duplications arise
A
Breaks
Unequal crossing over
18
Q
Duplications can be detected when
A
Meiosis
19
Q
Consequences of deletions
A
- Redundancy
- alter gene dosage
- pseudogenes
- copy acquires a new function
20
Q
Inversions
A
Two breaks on a chromosome followed by reinversion in the opposite orientation
21
Q
Two types of inversions
A
- Peri-centric inversions
2. Para-centric inversions
22
Q
Pericentric inversions
A
Two breaks span the centromere
23
Q
Para-centric inversions
A
Two breaks on one arm of the Chromosome
24
Q
Consequences of inversions on phenotype
A
- Reverses orientation
- many function in same or different way
- like the white eye gene drosophila
25
Consequences for recombination and production of gametes from inversions
- Usually viable if no recombination occurs
| - can be viable or non-viable if crossing over occurs
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Two results of paracentric inversions
- dicentric chromatid
| - Accentric chromatid
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Dicentric chromatid
2 attached, missing large segment
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Accentric chromatid
Lost piece from dicentric
- no viable gametes
- Reduced fertility
- reduced recombination frequency
29
Results of pericentric inversions
- Reduced recombination
- reduced fertility
- some viable gametes
30
Translocation causes…
- Exchange of segments
- reciprocal or non-reciprocal
- if no genetic material is lost, it is a balanced translocation
31
Consequences of tranlocations
- can alter expression
- phenotype varies
- possible new gene products
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Philadelphia chromosome
Translocation between chromosomes 9 and 22
33
Aneuploidy
Increase or decrease in number of individual chromosomes
34
Polyploidy
Increase in the number of sets of chromosomes
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4 most common types of aneuploidy
- nullisomy
- monosomy
- trisomy
- tetrasomy
36
Nullisomy
Loss of both members of a pair of homologous chromosomes
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Monosomy
Loss of a single chromosome
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Trisomy
Gain of a single chromosome
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Tetrasomy
Gain of two homologous chromosomes
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2 less common aneuploidy types
- double monosomy
| - double trisomy
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Origins of aneuploidy
- Non-dysfunction in meiosis or mitosis (failure of chromosomes to separate)
- deletion of a centromere leading to chromosome loss
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Outcomes of non-disjunction aneuploidy
Trisomy- may be visable
| Monosomy- usually not visable
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Common aneuploidy
- Trisomy 13- edwards syndrome
- Trisomy 18- Patra sybrome
- Trisomy 21-down syndrome
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Sex aneuploidy
Monosomy - 1 X- Turner syndrome
| extra X copies - klinefeller syndrome
45
Primary Down syndrome
- Trisomy 21
- most cases
- non disjunction
- mother contributes extra chromosome
- usually happens with mother’s age
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Familial Down syndrome
- 3rd chromosome is attached to another chromosome usually 14 or 15
- 3-4% of cases
- translocation carrier is one parent
47
Polyploidy
More than 2 sets of chromosomes
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Polyploidy is common in
Plants and is important in plants
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Two types of polyploidy
1. Autopolyploid
| 2. Allopolyploid
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Autopolyploid
Multiples of the same genome
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Allopolyploid
Multiples of closely related genome
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Origins of autoploidy
- Mitosis or meiosis
| - non-disjunction of all chromosomes
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Effects of autoploidy
- Usually sterile
| - most gametes produced are genetically unbalanced
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To convert sterile hybrid into new fertile species
- hybrid is sterile
- unbalanced gametes are non-viable
- but if entire genome is doubled by mitosis non-disjunction, fertility problem is solved
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Significance of polyploidy
Agriculture
56
What makes viable hybrid
Mitotic nondisjunction events
| -cabbish
57
Restriction Endonucoeases
Recognize specific DNA sequences and restrict entry of foreign DNA
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Most important type of RE’s in biology
Type 2
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Types of restriction enzymes
EcoRI
| Etc
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Process of the RE’s
- Sticky ends when it’s EcoRI
- ligament glued them back together
- ligament completes phosphodiester bond
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Why don’t RE’s destroy themselves
Host marks genome with methyl groups
62
Gel electrophoresis
Sorting DNA fragments by size
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DNA molecules have what kinda of charge under neural PH
Negative
64
Preparation of agarose gel
1. gel tray
2. prepare barriers
3. Pour molten gel in tray
4. Insert comb for wells, remove once set
5. Load DNA sample into gels
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Dye that is used so DNA can be seen
Common EtBr but now is a green1 or something like that
66
Migration rate is direction proportional to….
The log of it’s molecular mass
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What is important when using agarose gel
Concentration
68
Slowest DNA molecule based on topology
Circular
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Middle speed DNA molecule based on topology
Linear
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Fastest DNA molecule based on topology
Supercoiled
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What does not effect migration rate
GC content
72
Applications of qPCR
- not good at determining abundance
- end point is always directly related to starting point
- exponential phase exists but cannot be detected
- log makes the DNA linear
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Stages of qPCR data
- exponential
- linear
- Plateau
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qPCR can be used to measure…
The rate at which a gene is transcribed
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Minimum requirement for DNA synthesis
- template DNA strand
- primer
- DNA polymerase
- dNTP’s
- Mg+
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Mollisis’s insight
Enzymatic copying of DNA leads to qPCR
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Stages of PCR replication
- Denaturing
- annealing
- extension
- repeat
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Temperature of denaturing
95
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Temperature of annealing
50-65
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Temperature of extension
72
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PCR ingredients
- dNTP’s
- Mg+
- Primers
- Template DNA
- Thermostable DNA (Taq)
- salt, PH control, stablers
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PCR primers length
18-25bp
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Proper direction do primers
Primers extend from their own 5’-3’ ends so that’s how they’re set up on opposite sides
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Most PCR product is (size)
2Kb or less
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Why are primers 18-25bp long
- specific primers
- less expensive then larger ones which are more specific
- smaller would have more places to bind and would be less specific
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Applications of PCR in studies
- Amplifying target sequences
| - detection of rare DNA sequences, which has many applications in animals bio, forensics, and environmental stuff
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What is PCR not good at
Determining abundance’s
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Sanger method
- one of two DNA sequencing methods in 1970’s
- still used today
- remains good standard
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Minimum requirements for synthesis in vitro
- primer
- polymerase
- template
- dNTP’s
90
What is different about ddNTP’s
Lack 3’ OH group and dNTP’s cannot bind
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About fluorescent dideoxy sequencing
- labels with a fluorescent dye
| - a laser lights up the dyes and you can tell which dNTP or ddNTP is present
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Pros of Sanger dideoxy sequencing
- very accurate
- long reads Up to 1000bp
- easy, can be automated
- low cost
- continues to be used
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Cons of Sanger dideoxy sequencing
- too slow for genome sequencing
- costly when lots of data
- requires purification and preparation
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About the human genome project
- incredibly expensive
- Sanger method was used
- human genome now costs less than 1000$ to sequence
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Illumina DNA sequencing
- adapters are added to ends of DNA strands
- DNA is added to flow cell
- bridge amplification
- creates double strands, then denatures and results in clusters
- dNTP’s are then added
- laser excited dNTP base added after it attaches to matching base from clusters
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Other key points about illumina
- after dNTP’s are added they can still be replicated
| - millions of distinct DNA sequences determined simultaneously
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Nanopore sequencing
- single molecule at a time
- Enzyme unwinds DNA and strand is pulled by an electrical current through a pore membrane
- each base produces characteristic disturbance in electrical current
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Pros of nanopore sequencing
- long reads but less than Sanger
- no PCR step before sequencing
- small and portable
- used for rapid results
- may eventually be able to distinguish methylated based
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Cons of nanopore sequencing
-low accuracy compared to other methods but it is getting better
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Illumina and nanopore are both
Much cheaper than Sanger method
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Applications of genome assembly by shotgun sequencing
- short sequences of genomic DNA
- sequenced by next gen
- assembly software looks for sequence overlaps
- currently preferred way of sequencing genomes but has problems with repetitive sequences
- nanopore is used to over come above problem
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Nanopore by itself is not great for
Genome sequencing
103
Application of transcriptiomics (gene expression analysis)
- isolate mRNA
- convert to cDNA
- shear cDNA, add adaptors
- sequenced by next gen
- bioinformatics software sorts sequences into different types of genes
- number of types each gene appears measures its expression
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DNA barcoding is used for…
- fish DNA labeling
- shark fin trade (endangered species)
- evolutionary relationship of species
- studying microbiomes
- environmental DNA
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How DNA barcoding is done
- isolate DNA from sample
- amplifying microbial sequences using 16s rDNA primers
- sequence using next gen
- run data through databases
106
Why study genetic variation at the molecular level
- determine genetic basis of inherited diseases or phenotypic traits
- study relatedness of individuals
- to identify individuals
- percentage analysis or inferring pedigrees
- identify criminals
107
DNA fingerprinting
-minisatilite DNA, Tandem arrays, same sequence repeated for about 10-100bp
108
DNA profiling is now done with…
Microsatilites
109
About microsatilites
- shorter repeating units 5-10bp
- more specific
- can be amplified by PCR
- can be called short tandem repeats (STR’s or SSR’s)
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Microsatilite genotyping
- PCR primers designed for flanking sequences
- primers are fluorescently labeled
- amplify products of different sizes
- separate by electrophoresis
- genotypes identified by size of products
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Codominant detection
- Heterozygote produce 2 bands, means both alleles are detected
- usually uses same capillary electrophoresis machines used for dideoxy sequencing
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Does STR use fluorescent colouring
Yes, the primers are labeled
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How is STR used in forensics
13 loci detect enough variability to distinguish individuals and they are all widely used
114
Other methods STR is used in forensics
- PCR based microsatilite genotyping requires only small amounts of DNA
- DNA based evidence convicts and exonerates people
- these methods are sensitive and contamination can be problematic
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A few microsatilites cause genetics disorders
- they generally occur outside of Exxon’s
- trinucleotide repeats
- myotonic dystrophy
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Use of RE’s to detect DNA polymorphism
- mutations create or destroy restriction endonuclease sites
- gain or loss of restriction sites detected using electrophoresis
- restriction site polymorphisms are commonly caused by nucleotide polymorphisms
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RFLP
Restriction fragment length polymorphism
118
SNP
Single nucleotide polymorphism
119
SNP’s
- SNP single base mutations are most common genetic variations
- an SNP occurs every 800-1000bp
- any two humans will have several million SNP loci
- usually di-allelic
- current technologies allow SNP’s to be genotypes simultaneously
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What is a haplotype
-arbitrarily long stretch of DNA charactorised by particular allele at SNP positions on the sequence
121
SNP Chips
Are used to genotype large numbers of SNP’s, allowing many to be done at once
122
CRISPR
Clustered regularly spaced palendromic repeats
123
About CRISPR
- originally discovered 1987
- designed to target specific DNA molecules comparable to vertebrae adoptive immune system
- many CRISPR-Cas systems
- found in 50% of bacterial species and 90% of archea
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CRISPR-Cas9
1. cas gene encodes CRISPR-associated proteins
| 2. CRISPR contains unique spacer DNA from invading phage between short planedromic repeat DNA sequences
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Three stages of defence against foreign DNA
1. Foreign DNA acquisition
2. expression
3. Interference
126
Stage 1: foreign DNA aqquisition
- invading bacteriophage injects foriegn DNA into cell
- foreign DNA is cut into short segments
- DNA segments are added into bacterial Chromosome between repeated palindromic sequences
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Stage 2: expression
- case genes transcribed and translated to proteins
- CRISPR locus transcribed to pre-crRNA
- note how palindromic sequences fold back to hairpins
128
Stage 3: interference
- crRNA is incorporated into cas protein and allow targeting to DNA sequences that match the unique spacer rna
- recognizes DNA sequences that is encountered before
- binds and used cas endonuclease activity to clone invading dna
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Closer look at cas effector
- prescience of Protospacer adjacent motif is required in target DNA
- a PAM is a short consensus sequence
- transactivating small RNA (tracrRNA)
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The key innovation
Substitution of chimeric gDNA in of natural crDNA and tracrRNA
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Editing genes with CRISPR-cas9 step 1
- sgRNA is designed to target a specific sequence
- sgRNA assembles with cas9 protein to form effector complex
- effector complex first finds Pam, then cas9 unwinds DNA immediately upstream
- if target sequence is present 20b5’ end of sgRNA binds with it
- cas9 makes double stranded cut in genome
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Gene editing with crispr cas9 step 2
-cellular DNA repair mechanisms engaged, with type possible outcomes
133
2 possible outcomes of cellular repair after step 2 of genome editing with CRISPR cas9
1. Broken ends can be rejoined without any template
| 2. Broken ends can be rejoined with template
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Non-homologous end joining (NHEJ)
- most common type of repair to double stranded breaks
- no template used
- nucleotides may be randomly inserted or deleted as cleaved ends rejoin
- often results in insertions or deletions
- frameshifts lead to non-functional alleles
135
Homologous directed repair (HDR)
- another way to repair double stranded breaks in DNA
- uses same repair enzymes as in crossing over or recombination
- can use a homologous chromosome as a template
- in CRISPR excitements can inject donor DNA or guide the CRISPR-cas9 to stimulate HDR
- can add new sequences
136
Advantages of CRISPR-cas9
- cheap and easy
- targeting
- relatively specific
- indels created by non-homologous end joining to create gene knockouts to determine function phenotype
- can be introduced to intact, living cells
- can introduce cas9 with donor DNA to stimulate HDR
137
Challanges of CRISPR-cas9
- off targeting
- can be hard to control if NHEJ or HDR is used
- mosaicism: not all cells edited, differen genomes get different effects
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Potential uses of crispr mediated gene editing
- basic research, disrupt gene to determine function
| - editing genomes to meet human needs like pig livers or farm animals for best food
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Reasons for cloning genes, gene transcripts and DNA (living)
- to make more DNA with high fidelity for further study or manipulation
- to produce substances o scientific or commercial value
- to modify genome of plants or animals to introduce new desired traits
140
Cloning requires a vector
-plasmid is commonly used
141
Plasmids contain
- origin of replication
- selectable markers to identify cells that have taken up plasmid
- unique restriction enzyme cleavage sites
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Puc19
- Typical and commonly used bacterial vector
- contains a portion of lacZ+ with unique restriction enzyme cut sites
- antibiotic restriction enzyme (ampR)
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Inserting foreign DNA sequences into a plasmid
- cut foreign DNA with a restriction enzyme
- cut plasmid with the same restriction enzyme or one that provides comparable fragment ends
- mix cut foreign DNA and cut plasmid DNA
- use DNA ligase to seal sugar phosphate bonds
144
Transforming bacteria and selecting for recombinants
- competent bacteria: Ecoli made receptive
- lacZ- doesn’t have the lacZ portion present in plasmid
- lighted plasmids containing DNA inserts are used to transform competent cells
145
Part 2 transforming bacteria
- transformed bacteria are plated out in agar
- bacteria with no plasmid does not grow
- bacteria with non-recombinant plasmid: produces B-galactidase and blue colonies
- bacteria with recombinant plasmid: does not produce b-galactidase and has white colonies
146
Bacterial expression vectors
- include opening and regulatory sequences to allow expression of genes in bacteria
- good for production of many enzymes, especially those that originate from bacteria
- not good for gene products that require post-transcriptional modification, occurs with many eukaryotic proteins
147
Use of plasmid vectors to transform plans and fish
-can be co-opted to introduce new genes into plants
148
Molecular toolbox
- RE’s repurposed as DNA scissors
- ligases allow DNA molecules cute by RE’s to be recombined with plasmids and vectors that transform organisms
- PCR competent to make modest amount of a petticoat sequence
- cloning is best when large amounts are needed
- PCR is also used to check success of experiments
- gel electrophoresis is used routinely in combination with RE cleavage or PCR to check the success in cloning or transfusion experiments
