Lab exam Flashcards
Karyotype
# and appearance of chromosomes in a eukaryotic cell Also used for the complete set of chromosomes in a species, or an individual organism
Karyotypes can also be used to study ____.
Chromosomal abberations
P arm in the human chromosome is _____.
Short
Q arm in a human chromosome is _____.
Long
HeLA
A human tumor cell line named after Henrietta Lacks
Aneauploid
1 or more whole chromosomes of a normal set of chromosomes that are missing or are present in more than the usual number
Chromosome types:
Metacentric, Sub-metacentric, and acrocentric
Colchicine
A plane alkaloid that arrests cells in metaphase
Why is a HeLa cell arrested in metaphase?
Because it’s when the cell is most highly condensed, highly coiled and easiest to distinguish
Cells are ___ in order to get them to detach from the culture plate
Trypsinized
Why are HeLa cells exposed to a hypotonic solution?
It caused osmotic swelling which causes the membranes to rupture easily and spill out the chromosomes, so you can see the chromosomes spread
Eosin
Pink colour and stains nucleic acids (or the nucleus)
Haematoxylin
Purple colour and stains nucleic acids (or the nucleus)
GMO (Genetically modified organism)
Is an organism, plant, or animal that has been genetically manipulated
Disadvantages of GMO foods
- Less sugar, protein, fibre, zinc
- Exposure to possible toxins
- Harm to environment
- Antibiotic resistance
- Spread of introduced genes to non-target plants
What are the 3 stages of a polymerase chain reaction?
Denature, Anneal primers, Extend
Purpose of gel electrophoresis
To separate, identify, and purify DNA fragments
2 types of gel electrophoresis
Agorase, Polyacrylamide
Why use agarose gel?
Lower resolution, greater range or separation
-Runs in horizontal configuration under constant strength electric field
Why use gel electrophoresis?
- To determine if sample contains a specific segment of DNA
- Compare DNA samples
- Identify samples as a given species
- Isolate and sequence DNA
- Identify variances in DNA sequences
Which way does DNA flow in electrophoresis?
From negative to positive
-Because DNA is negatively charged
____ DNA fragments will migrate further down the length of the gel than ____ fragments
Smaller; Larger
Why put glycerol in well for gel electrophoresis?
Increases the density of sample which keeps DNA in the well
Why add bromophenol blue and xylene cyanol into wells for gel electrophoresis?
Dyes
-They track the migration of DNA sample in the gel
Xylene: -4000bp
Bromo: -300bp
What are factors that affect the rate of DNA migration in gel electrophoresis?
- Agarose concentration
- Applied voltage
- Ionic strength of buffer
- # of bp in sample
- DNA confirmation
- Size of gel
What is ethidium bromide?
Fluorescent dye used in gel electrophoresis
- Absorbs UV light
- Dangerous
Why is SYBR safe?
Same as ethidium bromide but not as toxic
Gene nomenclature
3 italicized lower case letters
Gene product nomenclature
First letter capitalized, no italics
Ex: LacZ
Operon
Structural genes under the control of 1 promoter
Lac promoter
RNA polymerase binds
Lac terminator
Gene that specifies end of transcription
LacZ
Encodes beta-galactosidase –> Causes lactose –> Galactose and glucose
LacY
Encodes lactose permease (Which transports lactose)
LacA
Encodes transacetylase
Competent cells are able to uptake exogenous ____.
DNA
E.Coli artificially induced to enter competency when treated with ____.
Metal cations- Ca 2+, Mg 2+
Non-coding regions of the genome contain ____.
VNTR
Variable number of random repeats
p < 0.05
Reject null hypothesis
p > 0.05
Fail to reject the null
assumption is correct
Epistasis
Genes that modify the expression of other genes
What is a meningioma?
Benign tumour that arise from meninges covering the brain and spinal cord
Somatic line mutation
A non-inheritable mutation occurring in a somatic cell
Germ line mutation
An embryonic cell lineage that forms that reproductive cells
Sporadic disease
Occurs in single and scattered cases
Familial disease
Transmitted through and expressed by members of a family
What is chromatin remodelling?
A process in which the structure of chromatin is altered by a protein complex
-Results in change in the transcriptional stage of genes
Define exome sequencing
Selective sequencing of the coding regions of the genome
Exons
Short, functionally important sequences of DNA
-Ones that are translated in proteins
Causes of meningiomas
NF2 mutations, Sporadic cases
How often are there germ line cases found in meningiomas?
Rarely
Positive control
Receives a treatment with a known response
Negative control
Groups where no phenomenon are expected
Define experimental control
A standard used for comparison that contains everything except the variable(s) you are testing
GM food lab: one control for the PCR
1 sample without DNA
Lac operon with glucose present
No cAMP production and no lac operon transcription
Define loss of heterozygosity
Loss of the wild type allele in a somatic cell
Define immunohistochemistry
- Antibody that binds to protein or another molecule that reacts to produce a visual stain
- Detects presence of protein
What does hemtoylin and eosin stain?
H-stains nucleus
E-counterstain, background is pink
Define clear cell histology
Cell whose cytoplasm is not stained by hematoxylin or eosin
-Indicates a neoplastic cell
How do you enable E.Coli to take up exogenous DNA?
- Treat with calcium chloride and heat
- DMSO increases solubility of lipids in outer membrane
- Ca 2+ creates positive charge on the membrane that will attract the negative DNA
Why did recovery broth lack ampicillin?
Have to give bacteria time to transcribe gene for ampicillin resistance
How would you differentiate between plasmids with gap in the correct (5’ to 3’) or incorrect orientation?
The gap and amp resistance gene are located on separate parts of the plasmid so the incorrect orientation of gfp wouldn’t affect resistance.
-These would still survive, but they wouldn’t fluoresce
Steps cell apoptosis
- Nuclear DNA fragmented
- Internal cellular structures are disrupted
- Cell dissolved into small spherical structures: apoptotic bodies
What happens when tumour-suppressor genes are mutated?
Cells are unable to respond to cell cycle checkpoints or do not undergo cell death
-Leads to accumulation or more mutations and development of cancer
What is optimal primer length?
18-24bp
What is optimal melting temp. for forward/reverse primers?
52-60 degree difference between the 2
What is optimal G/C content for primers?
45-55%
What is the optimal amplicon length of primers?
300-1000bp
Query cover
How much of the primer sequence is covered by alignment to the database sequence
E value
Probability of the sequence occurring the database
-Want it to be low
Identity
The extent to which 2 sequences have the same residues at the same positions in an alignment
Why boil with Chelex?
Boiling denatures proteins and Chelex protects sample from DNAases which cut DNA into small fragments making is unusable for PCR
What does Chelex need to bind to DNAases?
Mg 2+ (Magnesium Chloride)
dNTP’s
Deoxynucleotide Triphosphates
-Building blocks for DNA
How do you choose annealing temp?
5 degrees lower than the Tm
Describe initial denaturation in PCR
94 degrees for 3 minutes
-Heated to this temp in order to denature the double stranded DNA into single strands
What temperature for Extension in PCR? Why?
72 degrees
-Allows synthesis of new DNA
Temperatures for PCR
- Initial denaturation: 94 (3 mins)
- Denature: 94 (1 min)
- Anneal: 54 (1 min)
- Extension: 72 (1 min)
- Hold at 12 degrees
Optimal primer: There are G/Cs within the last ___ bases of the ___ end, but not more than ___ of either G or C.
4
3’
3
Mitosis
The process of nuclear division of either a diploid (2n) or haploid (n) eukaryotic cell whereby 2 daughter nuclei are produced that are genetically identical to the parent nucleus
-Cell division usually follows nuclear division
What happens in the 1st phase (G1, Gap 1) of the cell cycle?
Cell growth and prep for DNA replication
What are included in interphase?
G1, S, G2
What occurs in S phase?
DNA replication and chromatid duplication
What takes place in the G2 phase?
More cell growth
When is a single centrosome duplicated?
Early in interphase
Prophase
- Chromosome becomes visible
- Nucleolus disappears
- Spindles form
- Nuclear envelope disappears
Metaphase
Chromosomes arrange on the place and attach to spindle
Anaphase
Sister chromatids separate and move to opposite poles
Telophase
Chromosome sets assemble at opposite poles
- Nuclear envelope reforms around each set
- Cytokinesis follows usually
Cytokinesis
The cytoplasm divides, the cell contents are separated into 2 separate cells
S phase of telophase
Migration of the daughter chromosome s to the 2 poles is complete
How is the chromosome in G1 different from G2?
G1: Single and unreplicated
G2: Present as 2 chromatids
Meiosis
Process producing 4 haploid daughter cells from 1 diploid (2n) cell.
-2 successive divisions: Meiosis I and Meiosis II
What happens prior to meiosis?
All chromosomes are duplicated prior to this
Meiosis I: Prophase I
- Chromosomes become visible
- Crossing over occurs
- Nucleolus disappears
- Meiotic spindle forms
- Nuclear envelope disappears
When does crossing over of homologous chromosome s occur?
Prophase I
Meiosis I: Metaphase I
Pairs of chromosome become arranged on the metaphase plate and attach to fully formed spindle
Meiosis I: Anaphase I
Sister chromatids separate and migrate towards opposite poles
Meiosis I: Telophase I
Homologous chromosome pairs reach the poles of the cells
- Nuclear envelope forms
- Cytokinesis follows to produce 2 daughter cells
What does DNA replication occur?
S phase
When do sister chromatids separate?
Anaphase II
Meiosis II: Prophase II
Nuclear envelope breaks down and the spindle forms
Meiosis II: Metaphase II
Chromosomes becomes arranged on the plate and attach to spindle
Meiosis II: Anaphase II
The 2 chromosomes in each bivalent separate and migrate towards opposite poles
What is the stage of meiosis during which the pairs of chromosomes become arranged on a plane equidistant from the poles?
Metaphase I
What is the key difference between Anaphase I in meiosis and Anaphase in mitosis?
Sister chromatids remain joined during anaphase I in meiosis, whereas in anaphase of mitosis they separate
In what stage of meiosis do the centromeres separate, and the 2 chromatids of each chromosome move to opposite poles on the spindle?
Anaphase II
When is synapsis of homologous chromosomes?
Prophase I
Synapsis
Pairing of homologous chromosomes
When does the independent assortment of unlinked genes occur?
Metaphase I
When does the reduction of chromosome number in cell from 2n to n?
Telophase I
What 2 events in meiosis lead directly to genetic diversity?
Crossing over and independent assortment
Distinguish between homologous and non-homologous chromosomes
Homologous: Contain alleles for the same type of genes
Non-homologous: Contain alleles for different types of genes
Distinguish between chromatids and chromosomes
Chromatid is a single strand
Chromosomes consist of 2 sister chromatids
Wild type
Most abundant phenotype among individuals of a particular species
Mutant
A phenotype resulting from a mutation
-Anything other than the wild type
Map unit
An arbitrary unit of measure used to describe relative distances between linked genes
When calculating the map distance from the centromere why do you divide by 2?
Each crossover produces 2 spores like the parents and 2 new spores as a result of the crossover.
So only 2 are the result of crossing over.
Name the 3 types of chromosomes found in the HeLa cells based on the location of the centromere
Metacentric (P=Q)
Submetacentric (P
Klinefelter Syndrome
XXY
XXXY
XXXXY
Turner syndrome
X
Female with trisomy X
XXX
Trisomy 21, Trisomy 18, Trisomy 13 (Downs, Edwards, Patau)
XX or XY
Submetacentric
P
3 phases of PCR
Denaturation
Annealing of primers
Polymerization
At what temperatures do proteins usually start to denature?
Temperatures above 43 degrees
Why can taq polymerase withstand high temps?
Because it comes from bacteria that live in hot springs and hydrothermal vents
From which organism does Taq originally come from?
Thermus aquaticus
What is the potential use of PCR products?
Detect mutations
Recombine
Paternity tests
Solve a crime
What happens to PCR when you forget to add DNA?
No bands appear
Which were the positive controls for the soy PCR lab?
Plant chloroplast, Soy lectin
Both are plant and soy specific
What happens to PCR if you only add 1 primer?
Instead of a geometric amplification, you get a linear one
-Much less product
What is the function of the buffer in gel electrophoresis?
Prevents the anode and cathode from becoming alkaline and acidic
-Also contains ions to help transport the current
The rate of linear DNA migration is inversely proportional to _____?
The log of its molecular weight
*Does not include circular DNA
Function of transacetylase
Covalently modifies lactose and lactose analogues
How is lac operon regulated?
Negative control mechanism: Lac repressor protein binds to the lac operator site and prevents RNA polymerase from transcribing
Catabolic repression: cAMP binds to CAP. This complex binds to lac site and increases rate of transcription. Glucose decreases cAMP production.
Functions of beta-galactosidase
- Catalyzes the breakdown of lactose into glucose and galactose
- Catalyzes conversion of lactose into allolactose
Ampicillin
Derivative of penicillin and inhibits bacterial growth by interfering with the synthesis of cell walls
What is a DNAase?
Enzymes that occur naturally in all body tissues
-Cut DNA into small fragments rendering it unsuitable for PCR
What cause cranial and spinal meningiomas?
- Associated with a tumour suppressing syndrome
- Mutations in SWI/SNF chromatin-remodelling complex subunits which regulates gene transcription and apoptosis
Are individuals with familial spinal meningiomas associated with NF2 mutations?
No, they are not associated with these mutations
What does the loss of heterozygosity and SMARCE1 in tumours suggest?
That it functions as a tumour suppressor to induce apoptosis
What did the researchers use to screen the genome for mutation?
The researchers used exome sequencing for this
Describe how the loss of function mutations in SMARE1 predispose individuals to a specific disease phenotype
ie: spinal meningiomas
CNS tumours are rarely found in coffin-iris syndrome
- No one with spinal meningiomas has neurodevelopment problems
- So loss of function mutations in SMARE1 likely specific to familial spinal meningiomas
How does SMARE1 induce apoptosis?
Stimulates the expression of tumour suppressor genes
-Loss of activity leads to the cell not being triggered to undergo apoptosis
Trisomy 13
Patau syndrome
Trisomy 18
Edwards syndrome
Trisomy 21
Downs syndrome
