Introduction to cytogenetics Flashcards
(268 cards)
1
Q
What is clinical cytogenetics?
A
The branch of genetics that studies the relationship between chromosomal aberrations and pathologic conditions; the study of chromosome morphology, structure, pathology, function, and behaviour.
2
Q
What is molecular pathology?
A
A discipline that deals with the origins and mechanisms of diseases at the most fundamental level - macromolecules such as DNA and protein - in order to provide dx and discover possible avenues for rx.
3
Q
What is the difference between clinical cytogenetics and molecular pathology?
A
Low versus high resolution genetic analysis.
Cytogenetics analyses chromosomes at 1000X magnification and can detect changes (deletions/duplications) between 5 - 10 megabases (million bp) in size.
4
Q
What is the minimum size of genetic changes that can be detected under a microscope?
A
Changes that are 5 - 10 megabases (million base pairs) in length at 1000X magnification.
5
Q
How many base pairs are in the human genome?
A
3 billion.
6
Q
What is the minimum size of genetic changes that can be detected using molecular techniques?
A
A single base pair.
7
Q
What are the three main benefits of organising DNA into chromosomes?
A
- Packing large amounts of material into small spaces
- Less chance of breakage due to entanglement with other chromosomes
- Easier to separate during cell division
8
Q
How much does a DNA strand decrease in size by being packaged into a chromosome?
A
7000-fold decrease
9
Q
Which phase of the cell cycle is used for cytogenetics?
A
Metaphase (or prometaphase [border]).
10
Q
Which phases of the cell cycle make up interphase?
A
G1 (growth), S (synthesis [DNA replication]), and G2 (growth [preparation for mitosis]).
11
Q
What are the four main phases of mitosis?
A
Prophase, metaphase, anaphase, and telophase.
12
Q
What is the difference between mitosis and cytokinesis?
A
Mitosis is nuclear division, while cytokinesis is cellular division.
13
Q
What is chromatin?
A
Chromosomes of an interphase nuclei; decondensed chromosome
14
Q
What is the term for chromosomes of an interphase nuclei?
A
Chromatin
15
Q
What is the term for a decondensed chromosome?
A
Chromatin
16
Q
A non-dividing cell (G1) will have one chromosome consisting of one ____.
A
Chromatid
17
Q
A non-dividing cell (G1) will have one ____ consisting of one chromatid.
A
Chromosome
18
Q
What is replicated during S phase in a non-dividing cell?
A
The chromosome (consisting of one chromatid) so that the chromosome then consists of two sister chromatids.
19
Q
What is the difference between a chromosome of a non-dividing cell before and after S phase?
A
Before S phase the chromosome consists of one chromatid; after S phase the chromosome consists of two sister chromatids.
20
Q
The two sister chromatids of a non-dividing cell after S phase are bound at what structure?
A
The centromere
21
Q
What happens to the two sister chromatids of a chromosome following metaphase?
A
The sister chromatids separate and become daughter chromosomes (and are each referred to as chromosomes in their own right).
22
Q
Why is the classic ‘x’ form of chromosomes not visible when examining a metaphase cell under the microscope?
A
The sister chromatids are so closely bound that the gap cannot be seen; what looks like one chromosome under a microscope contains two chromatids.
23
Q
How many chromosomes are in a dividing cell?
A
46
24
Q
How many chromatids are in a dividing cell?
A
92
25
How many chromatids are in two paired chromosomes?
Four
26
How many chromosomes are in a gamete cell?
23
27
How many chromosomes are in a somatic cell?
46
28
Are diploid cells somatic or gamete cells?
Somatic
29
What is the meaning of 'diploid'?
Two copies of each chromosome
30
How many pairs of autosomes are in a somatic cell?
22
31
How many autosomes are in a somatic cell?
44
32
How many pairs of chromosomes are in a somatic cell?
23
33
How many sex chromosomes are in a somatic cell?
2
34
How many pairs of sex chromosomes are in a somatic cell?
1
35
Which type of chromosomes are labelled 1 to 22?
Autosomes
36
Which type of chromosomes are labelled 23?
Sex chromosomes
37
How many autosomes are in a gamete cell?
22
38
How many sex chromosomes are in a gamete cell?
1
39
Are haploid cells somatic or gamete cells?
Gamete
40
What is the meaning of 'haploid'?
One copy of each chromosome
41
Name the structure at both ends of a chromosome.
Telomere
42
What is a telomere?
The structure found at both ends of a chromosome.
43
What is the name for the short arm of a chromosome?
P arm
44
What is the name for the long arm of a chromosome?
Q arm
45
What are telomeres?
Regions of DNA at the ends of chromosomes that consist of TTAGGG repeats and are required for replication and stability.
46
Telomeres consist of ____ repeats.
TTAGGG
47
TTAGGG repeats are a feature of what chromosome structure?
Telomeres
48
How do telomeres protect chromosomes?
TTAGGG repeats are lost during each cell division which has a protective role in preventing end-to-end fusion and therefore promoting cell survival.
49
What is end-to-end fusion?
An open chromosome attaching to the end of another open chromosome, resulting in translocation or abnormality.
50
What is the centromere?
The constricted region in a chromosome where sister chromatids are held together, and the region where attachment to the mitotic spindle occurs.
51
What is the term for the constricted region where attachment to the mitotic spindle occurs?
Centromere
52
Which region is responsible for the accurate segregation of the replicated chromosomes to daughter cells during mitosis/meiosis?
Centromere
53
What is a metacentric chromosome?
A chromosome with a centromere in the middle (p and q arms are ~ the same length).
54
What is the term for a chromosome with a centromere in the middle (p and q arms are ~ the same length)?
Metacentric
55
What is a submetacentric chromosome?
A chromosome with a centromere positioned off the middle but not fully at one end (p arm is longer than the q arm).
56
What is the term for a chromosome with a centromere positioned off the middle but not fully at one end (p arm is longer than the q arm)?
Submetacentric
57
What is an acrocentric chromosome?
A chromosome with a centromere positioned at one end of the chromosome (leaves a satellite on stalks that is the p arm).
58
What is the term for a chromosome with a centromere positioned at one end of the chromosome (leavies a satellite on stalkds that is the p arm)?
Acrocentric
59
What is a karyogram?
A diagram/photograph of a cell with chromosomes ordered into pairs and in numerical order based on banding patterns.
60
What is the term for a diagram/photograph of a cell with chromosomes ordered into pairs and in numerical order based on banding patterns?
Karyogram
61
What is a karyotype?
A written description of a detected chromosome complement (e.g. 46,XY).
62
What is the term for a written description of a detected chromosome complement (e.g. 46,XY)?
Karyotype
63
List the four pathogenic mechanisms by which chromosome abnormalities result in a pathogenic cytogenetic abnormality (affect normal function).
1. Dosage effect
2. Direct damaging effect
3. Position effect
4. Imprinting
64
Describe the 'dosage effect' pathogenic mechanism of cytogenetic abnormality.
Lack or excess of chromosomal material (which directly corresponds to the amount of protein and gene expression).
65
What is the term for a pathogenic mechanism of cytogenetic abnormality caused by lack or excess of chromosomal material?
Dosage effect
66
Describe the 'direct damaging effect' pathogenic mechanism of cytogenetic abnormality.
Disruption of a gene that causes dysfunctional/non-functional genes.
67
What is the term for a pathogenic mechanism of cytogenetic abnormality caused by disruption of a gene that causes dysfunctional/non-functional genes?
Direct damaging effect
68
Describe the 'position effect' pathogenic mechanism of cytogenetic abnormality.
A gene in a new position functions inappropriately.
69
What is the term for a pathogenic mechanism of cytogenetic abnormality caused by a gene in a new position functioning inappropriately?
Position effect
70
Describe the 'imprinting effect' pathogenic mechanism of cytogenetic abnormality.
Unequal parental origin of a chromosome causing some of the chromosomes/DNA to be silenced.
71
What is the term for a pathogenic mechanism of cytogenetic abnormality caused by unequal parental origin of a chromosome causing some of the chromosomes/DNA to be silenced?
Imprinting effect
72
What is a constitutional abnormality?
A chromosome abnormality that is present from contraception and usually affects all cells of the body.
73
What is the term for a chromosome abnormality that is present from contraception and usually affects all cells in the body?
Constitutional abnormality
74
Infertility, problems of early growth and development, and family history complications are usually due to what kind of abnormality?
Constitutional abnormality
75
Constitutional abnormalities commonly cause what three issues?
Infertility, problems of early growth and development, and family history complications.
76
What kind of specimen should be collected when investigating constitutional abnormality?
Peripheral blood (from a whole blood sample).
77
What kind of specimen should be collected when investigating stillbirth/neonatal death (miscarriage)?
Products of contraception (POC - chorionic villi [contains foetal DNA and maintains its health well after foetal demise], amniotic sac, umbilical cord, foetal parts).
78
When are products of contraceptions usually taken?
In the first trimester which is when most large cytogenetic imbalances will cause pregnancy loss.
79
What kind of specimen should be collected when investigating the chromosome complement of a living foetus?
Amniotic fluid or chorionic villi.
80
What procedure is performed to obtain amniotic fluid for cytogenetic investigation of a living foetus?
Amniocentesis
81
What is amniocentesis?
A procedure in which a large needle is used to transabdominally aspirate amniotic fluid for cytogenetic analysis.
82
What are the limitations of amniocentesis for cytogenetic investigation of a living foetus?
- Can only be performed from 14 - 18 weeks gestation (~somewhat advanced)
- The cells in the fluid have been sloughed off by the foetus and are usually already dead so grow poorly in culture and contain poor DNA
83
What is chorionic villus sampling (CVS)?
A produce in which a large needle is used transabdominally or transvaginally to remove chorionic villus for cytogenetic analysis.
84
What are the advantages of chorionic villus sampling (CVS)?
- Can be collected from 10 - 12 weeks gestation
| - The cells are healthy so grow well in culture and yields better DNA
85
What is the risk that amniocentesis or chorionic villus sampling (CVS) will cause miscarriage?
0.5 - 1%
86
Are amniocentesis and chorionic villus sampling (CVS) a normal component of prenatal care?
No.
Amniocentesis and CVS are invasive sampling techniques that have a 0.5 - 1% chance of inducing miscarriage so are not performed unless indicated.
87
Amniocentesis and chorionic villus sampling (CVS) are both procedures guided by ____.
Ultrasound
88
What three kinds of specimen should be collected when investigating neoplasias (predominantly leukaemias)?
Peripheral blood, bone marrow aspirate and trephine, and solid tumours (e.g. tumours, lymph node, skin).
89
How long does mitosis typically take?
One hour
90
In which stage of mitosis do replicated chromosomes condense to form identifiable chromosomes?
Prophase
91
At which stage of mitosis does the nuclear envelope break down?
Prophase
92
At which stage of mitosis do spindle fibres form from the centrioles at each end of the poles?
Metaphase
93
What occurs in prophase?
Replicated chromosomes condense to form identifiable chromosomes and the nuclear envelope breaks down.
94
At which stage of mitosis do the chromosomes line up at the metaphase plate?
Metaphase
95
What occurs in metaphase?
Spindle fibres form from the centrioles at each end of the poles and the chromosomes line up at the metaphase plate.
96
At which stage of mitosis do the spindle fibres pull the sister chromatids to opposite poles and begins to form two daughter cells?
Anaphase
97
What occurs in anaphase?
The spindle fibres pull the sister chromatids to opposite poles and begins to form two daughter cells.
98
At which stage of mitosis is the nuclear envelope reformed?
Telophase
99
What occurs in telophase?
The nuclear envelope is reformed.
100
What is the purpose of cell culture and synchronisation?
To maximise chromosome length and the number of cells simultaneously in metaphase.
101
What are the two broad methods for culturing cells?
Cell suspensions and monolayer cultures.
102
Which broad type of culture is used for blood and bone marrows?
Cell suspensions
103
Which broad type of culture is used for amniotic fluids, CVS, tissues (not including bloods or bone marrows), and some tumours?
Monolayer cultures
104
What types of tissue are cell suspensions used to culture?
Bloods and bone marrows
105
What types of tissue are monolayer cultures used to culture?
Amniotic fluids, CVS, tissues (not including bloods or bone marrows), and some tumours.
106
What determines how successfully a culture has maximised the number of cells in metaphase?
Mitotic Index (MI)
107
What is the Mitotic Index (MI)?
The ratio of interphase to metaphase cells in a culture.
MI determines how successfully a culture has maximised the number of cells in metaphase.
108
What is the term for the ratio of interphase to metaphase cells in a culture?
Mitotic Index (MI)
109
A high Mitotic Index (MI) means that there are a lot of cells in ____ in that culture.
Metaphase
110
A ____ Mitotic Index (MI) means that there are a lot of cells in metaphase in that culture.
High
111
A low Mitotic Index (MI) means that there are a lot of cells in ____ in that culture.
Interphase
112
A ____ Mitotic Index (MI) means that there are a lot of cells in interphase in that culture.
Low
113
List the seven factors required for cell culture.
- Nutrients
- Hydration
- Warmth
- Respiration
- Waste removal
- Protection from pathogens
- Protection from physical harm
114
List the three properties of base media.
- Nutrients, vitamins, trace minerals
- Microenvironment
- Buffers pH changes
115
List the three components of complete media.
- Base media
- Growth serum
- Antibiotics
116
What is commonly used as growth serum in complete media?
Foetal calf serum (contains growth factors which promote cell division).
117
Which antibiotics are commonly used in complete media?
Penicillin and streptomycin.
118
Base media, growth serum, and antibiotics are the three components of what substance?
Complete media
119
What is marrowmax?
A commercial complete media commonly used for bone marrow cell culture.
120
Name a commercial complete media that is used for bone marrow cell culture.
Marrowmax
121
What is amniomax?
A commercial complete media commonly used for amniotic fluid cell culture.
122
Name a commercial complete media that is used for amniotic fluid cell culture.
Amniomax
123
What is PB max?
A commercial complete media commonly used for peripheral blood cell culture.
124
Name a commercial complete media commonly used for peripheral blood cell culture.
PB max.
125
What are the incubation conditions for normal peripheral blood cultures?
37'C
5% CO2
~72 hours
126
Why are peripheral blood cultures incubated at 5% CO2?
The CO2 reacts with the buffers in the media to keep the pH under control.
127
What cell type is the goal of culturing peripheral blood?
T cells
128
What is a mitogen?
A substance that stimulates cells to enter cell division.
129
What is the most common mitogen used to stimulate T cell division for constitutional blood work?
Phytohaemagglutinin (PHA)
130
What is phytohaemagglutinin (PHA)?
The most common mitogen used to stimulate T cell division for consitutional blood work.
131
Which mitogen can be added to oncology cultures to stimulate T and B cell proliferation?
Pokeweed mitogen
132
What is pokeweed mitogen?
A mitogen that can be added to oncology cultures to stimulate T and B cell proliferation.
133
Name the two DNA complements that work together and target B cells, stimulating B cell division and preventing apoptosis.
Interleukin-2 (IL-2) and DPS30
134
What are interleukin-2 (IL-2) and DSP30?
DNA complements that work together and target B cells, stimulating B cell division and preventing apoptosis.
135
A cell culture fixed at random would yield a very ____ MI.
Low
136
A cell culture fixed at random would show a slide full of cells in which phase of mitosis?
Interphase
137
What is synchronisation?
The process of getting or keeping most cells in a culture into metaphase.
138
Name the two techniques of synchronisation.
Block and release.
139
What is blocking (synchronisation)?
Addition of a reagent which blocks the cell cycle from proceeding past a specific stage, causing dividing cells to accumulate at the same mitotic point.
140
Name the technique in which a reagent is used to prevent the cell cycle from proceeding past a specific stage, causing dividign cells to accumulate at the same mitotic point.
Blocking, a synchronisation technique
141
Name a blocking reagent commonly used in cytogenetics.
Thymidine
142
How does thymidine work as a blocking reagent?
Thymidine blocks DNA synthesis, causing the cell cycle to slow to a near halt at the G1/S boundary.
143
What is releasing (synchronisation)?
Addition of a reagent which releases cell from blocking and allows them to resume division in a similar phase to other cells in the culture.
144
What is done to continue synchronisation when the majority of cells have reached the blocked stage of the cycle?
Addition of a releasing agent to resume the cell cycle with most cells in a similar phase.
145
What reagent is used to release a thymidine block?
2-dioxycytidine
146
What is 2-deoxycytidine used for?
Releasing a thymidine block.
147
What happens after 2-deoxycytidine is added?
The cells resume the cell cycle in a synchronised fashion.
148
What happens to cells during harvesting?
Cell swelling to make them brittle, fixing of cells in their current state, and removing unwanted cells (RBCs).
149
Why are RBCs removed during harvesting?
RBCs have no nucleus and no DNA so do not contain chromosomes to analyse.
150
What is the aim of harvesting cells for cytogenetics?
To fix cells in metaphase and prepare them for spreading.
151
What are the three stages of harvesting?
- mitotic arrest
- hypotonic treatment
- fix cells
152
What is the purpose of mitotic arrest in cell harvesting?
To optimise chromosomes in the desired condensed state.
153
Optimising chromosomes in the desired condensed state is the purpose of which stage of harvesting?
Mitotic arrest, the first stage of harvesting.
154
What is the purpose of hypotonic treatment in cell harvesting?
Swelling cells to disperse chromosomes and weakening the cell membrane to facilitate spreading.
155
Swelling cells to disperse chromosomes and weakening the cell membrane to facilitate spreading is the purpose of which stage of harvesting?
Hypotonic treatment, the second stage of harvesting.
156
What is the purpose of fixing cells in cell harvesting?
Preserve cells in their current state, hardening membranes and chromatin.
157
Preserving cells in their current state, hardening membranes and chromatin is the purpose of which state of harvesting?
Fixing cells, the third stage of harvesting.
158
What is used to optimise mitotic index (MI) and arrest cells in metaphase?
A spindle inhibitor (examples: colchicine and colcemid).
159
Colchecine and colcemid are examples of what reagent?
Spindle inhibitor.
160
What is the purpose of a spindle inhibitor?
To block cells in the optimal state by inhibiting polymerisation of the mitotic spindle.
161
How do spindle inhibitors block cells in the optimal state?
Inhibiting polymerisation of the mitotic spindle.
162
Addition of a spindle inhibitor such as colchicine is part of which stage of harvesting?
Arresting cells in metaphase, the first stage of harvesting.
163
Additional of a spindle inhibitor such as colchicine is part of which stage of sample processing?
Harvesting.
164
What substance added in harvesting increases the number of metaphase cells at harvest?
Colchicine, a spindle inhibitor.
165
Why are cells synchronised by blocking and releasing rather than simply adding colchicine (spindle inhibitor)?
Long exposure to colchicine shortens chromosomes and kills cells.
166
What two factors have an inverse relationship when deciding how long to expose cells to colchicine?
Number of metaphases : chromosome length.
Increased time = increased metaphases but decreased chromosome length.
Decreased time = decreased metaphases but increased chromosome length.
167
Would chromosomes exposed to colchicine for 15 minutes be long or short?
Long
168
Would chromosomes exposed to colchicine for 1 hour be long or short?
Short
169
How does adding a hypotonic solution remove RBCs?
Influx of water through their semi-permeable membrane via osmosis causes them to lyse.
170
What type of solution causes RBCs to lyse?
Hypotonic
171
What effect does adding hypotonic solution have on lymphocytes during harvesting?
Swells lymphocytes which spaces out chromosomes and makes cytoplasm fragile (enables chromosome spreading).
172
What type of solution is added during harvesting to swell lymphocytes, spacing out chromosomes and making the cytoplsam fragile for chromosome spreading?
Hypotonic solution.
173
How long should cells be left in hypotonic solution during harvesting?
~10 minutes.
174
Which hypotonic solution is commonly used for harvesting?
0.075M KCl (potassium chloride).
175
Is 0.075M KCl a hypertonic or hypotonic solution?
Hypotonic
176
At what stage of harvesting is 0.075M KCl added?
Hypotonic treatment, the second stage of harvesting.
177
What temperature is hypotonic treatment conducted at?
37'C
178
Why would hypotonic treatment be conducted at 37'C?
Warming the solution to 37'C softens the cell membrane and speeds cell swelling.
179
What is done to soften the cell membrane and speed cell swelling during hypotonic treatment stage of harvesting?
Warming the solution to 37'C
180
What is the optimal exposure time to hypotonic treatment during harvesting?
15 minutes.
181
Exposing cells to hypotonic treatment for 5 minutes would result in what kind of cell spreading?
Under-spreading.
182
Exposing cells to hypotonic treatment for 1 hour would result in what kind of cell spreading?
Over-spreading.
183
Exposing cells to hypotonic treatment for 15 minutes would result in what kind of cell spreading?
Optimal spreading.
184
What substance is universally used as a cytogenetic fixative?
Carnoy's fixative (3:1 methanol : acetic acid).
185
A 3:1 solution of methanol to acetic acid is commonly known by what name?
Carnoy's fixative.
186
What is Carnoy's fixative?
A 3:1 solution of methanol to acetic acid that is universally used as a cytogenetic fixative.
187
Why should Carnoy's fixative be added to cells slowly?
The solution can be harsh on cells.
188
What is done after adding Carnoy's fixative for cell fixation during harvesting?
The culture is centrifuged and supernatant removed - this is repeated until the cell pellet is white and the supernatant is clear.
189
How many times should the culture be centrifuged and supernatant removed after adding Carnoy's fixative during harvesting?
Until the cell pellet is white and the supernatant is clear, usually ~3 fixes (repeats).
190
What is the aim of chromosome spreading?
To produce well-spread metaphases with minimal chromosome overlap whilst avoiding chromosome loss.
191
What is the primary factor that affects chromosome spreading?
Drying time
192
List six variables that can effect chromosome spreading.
- Drying time
- Cell concentration
- Drop height
- Wicking effects (drying slide edges)
- Localised humidity
- Additional fix
193
What effect does high humidity have on chromosome spreading?
The slide dries slower which can cause overspreading.
194
What effect does high temperature have on chromosome spreading?
The slide dries faster which can cause underspreading.
195
What happens to Carnoy's fixative over time?
Methanol evaporates, leaving a higher percentage of acetic acid in the solution.
196
What effect does an older preparation of fixative have on chromosome spreading?
Methanol evaporates and leaves more acetic acid in the solution which can cause overspreading.
197
What effect does fanning the slide have on chromosome spreading?
The slide dries faster which can cause underspreading.
198
What effect does a high cell concentration have on chromosome spreading?
Less room in solution can result in underspreading.
199
What effect does drop height have on chromosome spreading?
The higher the drop the faster it expands when it hits the slide.
200
What effect does wicking (drawing liquid away by drying the edges of the slide) have on chromosome spreading?
The more the slide is dried, the more underspreading will occur.
201
What effect does adding additional fixative to the slide have on chromosome spreading?
Additional fixative slows the drying, causing more spreading.
202
What is the purpose of banding and staining?
To digest and stand chromosomes to create distinct banding patterns that allow identification of individual chromosomes and detection of structural and numerical arrangements.
203
What is a metacentric chromosome?
A chromosome with a centomere in the middle and an even p:q ratio.
204
What is a submetacentric chromosome?
A chromosome with a centromere between the middle and an end and an uneven p:q ratio.
205
What is an acrocentric chromosome?
A chromosome with a centromere at one end with a large q arm and a very small p arm (satellites and stalks).
206
What is the first step of banding and staining?
Ageing
207
What is the purpose of ageing when banding and staining?
Ageing gives sharper contrast to banded chromosomes.
208
How are chromosomes aged when banding and staining?
Being heated at 90 - 95'C for 1 hour.
209
List three different banding techniques.
- High resolution banding (Giemsa [G] banding)
- Constituative heterochromatin (C) banding
- Nucleolar organising region (NOR) banding
210
What is the most common banding used in cytogenetics?
Giemsa (G) banding
Also accepted: GTG banding (G-bands by trypsin using giemsa).
211
What is constituitive heterochromatin (C) banding?
Banding that stains gene poor regions, typically the centromere.
212
When would constituitive heterochromatin (C) banding be used?
Investigating variants or abnormal dicentric (two centromeres) chromosomes.
213
What banding technique should be used when investigating variants or abnormal dicentric abnormalities?
Constituitive heterochromatin (C) banding.
214
You examine a slide and see chromosomes with darkly stained centromeres. What kind of banding has been used?
Constituitive heterochromatin (C) banding.
215
What is nucleolar organising region (NOR) banding?
Banding that stains chromosome satellites (acrocentric chromosomes) with active NOR genes.
216
When would nucleolar organising region (NOR) banding be used?
Investigating rearrangements involving satellites.
217
What banding technique would be used when investigating rearrangements involving satellites?
Nucleolar organising region (NOR) banding.
218
You examine a slide and see that the satellite regions of some acrocentric chromosomes are darkly stained. What kind of banding has been used?
Nucleolar organising region (NOR) banding.
219
What is GTG banding?
G-banding by trypsin using giemsa - the most common type of giemsa banding in which chromosomes are treated with trypsin prior to giemsa staining.
220
What does trypsin do when used in GTG banding?
Trypsin partially digests some of the chromosomal histones, relaxing the chromosome structure so making DNA stains more accessible.
221
Which reagent used in GTG staining partially digests chromosomal histones, relaxing chromosome structure so making DNA stains more accessible?
Trypsin
222
GTG banding darkly stains gene poor ____-rich regions.
AT
223
GTG banding darkly stains gene ____ AT-rich regions.
Poor
224
Does GTG banding stain gene poor AT-rich regions light or dark?
Dark
225
GTG banding lightly stains gene rich ____-rich regions.
GC
226
GTG banding lightly stains gene ____ GC-rich regions.
Rich
227
Does GTG banding stain gene rich GC-rich regions light or dark?
Light
228
When examining GTG banded chromosomes you realise a large part of a band is missing. Which is more likely to have a significant impact - a missing dark section or a missing light section?
A missing light section as these are gene-rich regions.
229
How would the bands appear on an under-trypsinised slide?
Faint and poorly defined.
230
How would the chromosomes and bands appear on an over-trypsinised slide?
Chromosomes would look wide and 'motheaten' with fuzzy banding.
231
What is a G positive band?
A dark band in a chromosome.
232
What is a G negative band?
A pale band in a chromosome.
233
What is resolution in banding and staining?
The number of G positive and negative bands.
234
List four factors that can impact resolution.
- Cell type
- Colchicine time
- Ageing
- Trypsin time
235
How is resolution measured?
Resolution in measured in bands per haploid set (bphs) which directly correlates with quality assurance (QA) level.
236
What is the target resolution/QA level?
QA 6 (550 bands)
237
What is the aim of chromosome analysis?
To find and examine quality metaphase chromosomes from slides via microscopy.
238
What does chromosome analysis via microscopy entail?
Detection of numerical and structural abnormalities, and recording details of the cells examined including karyotyping and counting cells.
239
What is the minimum number of cells per sample required for karyotyping?
5 cells per sample.
240
What is the minimum number of cells per sample required for chromosome counting?
10 cells per sample.
241
In the minimum 5 cells required for karyotyping, how many times much each chromosome must have all regions unobscured?
At least twice.
242
What type of abnormality is found by counting chromosomes in karyotype analysis?
Numerical abnormalities
243
What type of abnormality is found by cross-examining banding patterns between chromosome pairs (homologues) in karyotype analysis?
Structural abnormalities
244
How many karyotypes are analysed per patient before reporting?
Five (minimum).
245
What is an ideogram?
A diagrammatic representation of a chromosome.
246
What two things are ideograms particularly useful for?
- Comparison of sex chromosomes in males
| - Breakpoint determination (finding where breaks occurred in a chromosome)
247
What is the format of a written karyptype (ISCN)?
Number of chromosomes, sex chromosomes, abnormality (if any).
248
What is the karyotype of a normal/healthy male?
46,XY
249
What is the karyotype of a normal/healthy female?
46,XX
250
What is the karyotype of a female with an additional (aneuploid) chromosome 21 (trisomy 21/Down syndrome)?
47,XX,+21
251
What is the karyotype of a male with a deletion between 13q21.1 and 13q31.1?
46,XY,del(13)(q21.1q31.1)
252
What is the karyotype of a female with a translocation between 6q27 and 15q22?
46,XX,t(6;15)(q27;q22)
253
What is fluorescence in situ hybridisation (FISH)?
A technique to detect and locate specific DNA sequences on a chromosome by hybridising complementary fluorescently labelled DNA probes.
254
What is a probe (FISH)?
DNA sequences correspondign to a particular region of a chromosome and fluoresces when excited.
255
What is hybridisation (FISH)?
The process of combining two complementary single-stranded DNA molecules and allowing them to reform a single double-stranded molecule through base pairing.
256
List the seven steps of fluorescence in situ hybridisation (FISH).
1. Fix sample to slide
2. Apply FISH probe to slide
3. Co-denaturation
4. Hybridisation
5. Stringency wash
6. Apply DAPI and antifade
7. Excite probe with filtered UV light and examine under a microscope.
257
How is co-denaturation achieved (FISH)?
Heating the slide to 74'C for 2 - 5 minutes.
258
What is co-denaturation (FISH)?
Third step of FISH - separation of target and probe dsDNA into ssDNA.
259
What is hybridisation (FISH)?
Fourth step of FISH - reannealing sample DNA and with incorporated probe DNA.
260
How is hybridisation achieved (FISH)?
Incubating the slide at 37'C for 2 - 48 hours.
261
What is a stringency wash (FISH)?
Fifth step of FISH - washing in SSC buffer to remove unbound probe.
262
What will happen if the SSC buffer used for a stringency wash is too hot or the salt too weak?
The wash will remove bound and unbound probe, stripping the slide.
263
What will happen if the SSC buffer used for a stringency wash is too cool or the salt too strong?
The wash will not remove unbound probe from the slide.
264
What is done after a stringency wash (FISH)?
Application of DAPI and antifade.
265
What does DAPI do (FISH)?
DAPI lightly stains chromosomes.
266
What does antifade do (FISH)?
Antifade protects probes (fluorophores) from light.
267
Can interphase cells be used for FISH?
Yes, interphase cells show probe location in relation to other probes and allows enumeration of signals.
268
What is the benefit of using metaphase cells over interphase cells for FISH?
Metaphase cells show probe location in relation to other probes, allow enumeration of signals, AND show probe position on chromosomes.
