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GH Coarsework 2023 > Techniques 1 > Flashcards

Techniques 1 Flashcards

1
Q

What are numerical chromosome abnormalities

A
  1. Aneuploidy
  2. Polyploidy
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2
Q

What are structural chromosome abnormalities

A
  1. Deletions
  2. Duplications
  3. Translocation
  4. Inversion
  5. Insertion
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3
Q

Characterise polyploidy

A
  1. More than 2 haploid chromosome sets
  2. Example: triploidy = 3 haploid chromosome sets =69 chromosomes
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4
Q

Characterise Aneuploidy

A
  1. Gain or loss of one chromosome
  2. Example: trisomy = 46+1=47 chromosomes
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5
Q

Explain what happens during Non-disjunction in meiosis 1

A
  1. Two homologous chromosome fail to separate
  2. Results in trisomic or nullisomic embryos
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6
Q

Explain what happens during non-dysfunction in meiosis 2

A
  1. Two sister chromatids don’t separate
  2. Results in trisomic or monosomic embryos
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7
Q

Mechanisms resulting in polyploidy

A
  1. Digyny
  2. Diandry
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8
Q

Define digyny

A

Diploid ovum is fertilised by a haploid spermatozoa

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9
Q

Define diandry

A
  1. Fertilisation of one oocyte by two spermatozoa.
  2. Four times more frequent that digyny.
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10
Q

Define mosaicism

A
  1. Occurs when there are two or more different cell lines present.
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11
Q

Define chimerism

A
  1. Two or more cell lines present originating from two or more zygotes
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12
Q

Name of the trisomy 13

A

Palau syndrome

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13
Q

Clinical features of Down syndrome

A
  1. Characteristic dystrophic features
  2. Intellectual delay
  3. Global development delay
  4. Hypotonia
  5. Congenital heart defects
  6. Hearing loss
  7. Vision problems
  8. Hypothyroidism
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14
Q

Clinical feature of Palau syndrome

A
  1. Severe intellectual disability
  2. Severe global development delay
  3. Congenital abnormalities of various organ systems
  4. Holoprosencephaly
  5. Polydactyly
  6. Cleft lip and/or palate
  7. Failure to thrive
  8. Poor prognosis
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15
Q

Name for trisomy 18

A

Edwards syndrome

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16
Q

Clinical features for Edward syndrome

A
  1. Severe intellectual disability
  2. Severe global developmental delay
  3. Microcephalic
  4. Clenched fists with overlapping digits
  5. Rocker bottom feet
  6. Congenital malformations of various organ system
  7. Poor prognosis
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17
Q

Turner syndrome is caused by

A

Missing X chromosome - 45,X

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18
Q

Clinical features of Turner syndrome

A
  1. Short stature
  2. Dysmorphic puberty
  3. Delayed puberty
  4. Infertility
  5. Lymphoedema
  6. Kidney abnormalities
  7. Cardiac abnormalities
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19
Q

Cause of Klinefelter syndrome

A

Males with an extra X chromosome - 47 XXY

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20
Q

Clinical feature of Klinefelter syndrome

A
  1. Tall stature
  2. Reduced muscle tone
  3. Small testes
  4. Delayed pubertal development
  5. Decreased facial and body hair
  6. Gynaecomastia
  7. Speech and language delays
  8. Behavioural problems
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21
Q

Characterise deletions

A
  1. Part of the chromosome is missing or deleted
  2. Some genetic material is missing can cause syndrome features, this depends on the genes involved in deletion.
  3. Can be sporadic or inherited
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22
Q

Different types of deletions.

A
  1. Interstitial deletion ( in the middle)
  2. Terminal deletion ( at the ends )
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23
Q

Example of diseases caused by deletions

A
  1. Wolf-hirschhorn syndrome -4p deletion
  2. Cri du chat syndrome - 5p deletion
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24
Q

Characterise Duplication

A
  1. Part of the chromosome is duplicated
  2. Results in extra genetic material, severity depends on the genes involved.
  3. Often less severe than deletions
  4. Can be. Sporadic or inherited
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25
Q

Diseases caused by duplications

A

22q duplication syndrome -22q11 duplication

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26
Q

Explain translocation ; reciprocal

A
  1. Exchange of maternal between chromosomes
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27
Q

What are balanced translocations

A
  1. Non loss or gain or genetic material.
  2. Individuals are usually phenotypically normal
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28
Q

What are unbalanced translocations

A
  1. Gain or loss of genetic material -severity depends on size
  2. Can be inherited from a balanced parent -recurrence is complicated
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29
Q

Explain what robertsonian translocation

A
  1. Exchange between two acrocentric chromosome to form mine large metacentric chromosome
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30
Q

Describe inversion

A
  1. Portion of a chronometer broke off, inverted and reattached.
  2. Can be Paracentric = on one arm
  3. Or pericentric = between the two chromosome
  4. If isolated the individual is often phenotypically normal
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31
Q

Describe insertions

A
  1. Proportion of a chromosome broke off and is inserted elsewhere.
  2. Interchromosomal = 2 different chromosomes
  3. intrachromosomal = within a single chromosome
  4. Direct insertion or inverted insertion
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32
Q

Characterise Ring chromosome

A
  1. Portion of a chromosome broke off and formed a ring
  2. Can happen with or without loss of genetic material
    3, very rare
  3. Often severe
  4. Usually sporadic
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33
Q

Characterise isochromosome

A
  1. Formed by a mirror image copy of a chromosome segment
  2. Results in a gain and loss of genetic material
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34
Q

Diseases caused by isochromosome

A
  1. Isochromosome 12 = pallister killan syndrome
  2. Turner syndrome =isochromosome Xq
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35
Q

Clinical indications for congenital cytogenetic prenatal testing

A
  1. Abnormal ultrasound findings
  2. Abnormal maternal serum screening
  3. Familial or parental chromosome abnormality
36
Q

Clinical indications for congenital cytogenetic postnatal

A
  1. Newborn/childhood growth and development problems.
  2. Adolescent/adult sexual development ad fertility. I.e amenhorrhea, history of miscarriages
37
Q

Clinical indications for molecular cytogenetic test FISH

A
  1. Micro-deletions/ duplication syndromes
  2. Confirm rearrangements suspected on a karyotype.
    3.Advance maternal age
  3. Birth defects or neural tube defects on ultrasound
38
Q

Clinical indications for molecular cytogenetic test microarray

A
  1. Unexplained multiple congenital abnormalities, dysmorphism, gross development delays, behavioural problems.
39
Q

Clinical indications for molecular cytogenetic test QF-PCR

A
  1. Rapid screening for common Aneuploidy chromosome.
  2. Advanced maternal age >35 years
  3. Sex determination (XX/XY)
40
Q

Function of L-glutamine (amino acid) in culturing

A
  1. Supplementary energy source for rapidly dividing cells.
  2. Serve as source of nitrogen for synthesis of proteins and nucleic acids
41
Q

Function of foetal bovine serum (FBS) in culturing

A
  1. Provide nutrients for cell growth
42
Q

Function of Antibiotics in culturing

A

Prevent microbial growth

43
Q

Function of phytohemagglutin (PHA)

A
  1. Mitogen which binds to the T-cells
  2. Thus stimulates cell division
  3. Resulting in mitotic cells
44
Q

Function of colcemid

A
  1. Arrests cells at prophase or early metaphase stage, while the chromosome are condensed and still intact.
45
Q

How does colcemid work.

A
  1. Binds to the spindle apparatus
  2. Preventing the formation of spindle fibres
  3. Resulting in spindle fibers not being able to attach to the centromeres
  4. Thus sister chromatids cannot be pulled into the opposite poles of the cells.
  5. Also stops the activity of formed spindle fibers.
46
Q

What is hypotonic treatment

A
  1. Process of increasing the cell volume
  2. In order to increase the cell surface area
  3. So that chromosome may evenly spread out
47
Q

Function of KCl/ sodium citrate

A
  1. Creates a concentration gradient across the cytoplasmic membrane.
  2. Thus allowing water to move from high concentration
  3. Resulting in the cell absorbing H2O and swelling
  4. Thus increasing the cell volume so that chromosome have sufficient space to spread out.
48
Q

Precautions and considerations of hypotonic treatment.

A
  1. Type of salt used can impact with width and sometimes the length of chromatids produced.
  2. Extended exposure to the hypotonic solution can cause the membrane to burst and allow some chromosome to escape.
49
Q

Explain the process of fixation.

A
  1. Lysine of erythrocytes and proteins.
  2. Preventing cells from further swelling
  3. Preserving the morphology of metaphase cells
  4. Hardening the cell membrane and chromatids thus preparing then for banding.
50
Q

Fixation of chromosomes in karyotyping is achieved by using?

A
  1. 3:1 methanol-acetic acid
  2. Kept in cold temperature
51
Q

Disadvantages of chromosomal analysis

A
  1. Requires fresh samples
  2. Prolonged culturing
  3. Prone to culture failure resulting in suboptimal or unsuccessful results.
  4. Microdeletions/duplications or cryptic rearrangements cannot be detected.
  5. Abnormalities caused by molecular genetic mutations cannot be detected.
52
Q

Making a slide depends on what variables.

A
  1. Height
  2. Air flow
  3. Slide quality
  4. Wet vs dry slide
  5. Angle of the slide
  6. Humidity / temperature
53
Q

What is aging during slide making

A
  1. Leads to cells becoming dehydrated.
  2. Thus allows metaphase cells to withstand further treatment during banding.
54
Q

What is differential banding and give examples

A
  1. Produces thin, alternating bands along the length of the entire chromosome.
  2. Examples: G-banding , R-banding, Q-banding
55
Q

What is selective banding and give examples

A
  1. Stains only a specific band or region.
  2. C- Banding and NOR staining
56
Q

Uses of G-banding

A
  1. Detection f both structure and numerical chromosome abnormalities
  2. Most commonly used banding method
57
Q

Uses of R-banding

A
  1. Detection of chromosomal rearrangements that involve the telomeres
58
Q

Uses of Q-banding

A
  1. Identifying the hetechromatin regions of certain chromosomes such as Y-chromosome and acrocentric chromosomes.
  2. Help to identify structural rearrangements involving Y-chromosome
59
Q

Uses of c-banding

A
  1. Identifying the number of centromeres present in a chromosomal rearrangement.
  2. Visualisation of constitutive heterochromatin at the centromeric regions of chromosome 1,9,16 and long arm of Y-chromosome.
60
Q

Use of Nor -staining

A
  1. Identify the stalk regions of acrocentric chromosomes especially when involved in translocations or in a marker chromosome.
  2. Confirm or exclude the presence of big satellites of acrocentric chromosomes
61
Q

Why would you do parentage testing.

A
  1. Asked for Magistrate/high court.
  2. Asked for by home affairs
  3. Private
  4. disease related
  5. Forensics
  6. Asked for by hospital
62
Q

Types of test done by DPL

A
  1. Trio : mother, child and alleged father
  2. Duo; father + child or mother +child
  3. Kinship: testing of a child and relative
63
Q

Why short tandem repeats area used in DPL.

A
  1. Repeat sequences of non-coding DNA
  2. Polymorphic
  3. Number of repeats is inherited
  4. Therefore, unrelated individuals unlikely to have the same number.
64
Q

What do DPL use for DNA extraction

A

Roche extraction kit

65
Q

Sample processing in DPL.

A
  1. Receiving
  2. DNA extraction
  3. Amplification
  4. Capillary electrophoresis
  5. Analysis of results
  6. Reporting
66
Q

Explain PREGEM Extraction steps.

A
  1. Remove one 3mm disc from the card-stored blood sample and place into a thin-walled PCR tube or a 96-well tray. For the best results, punch in the centre of the area where the blood was applied.
  2. Wash the disk in 100 microL of dna -free water by incubating at room temperature for 15 min. Aspirate the water from the disc and discard.
  3. Add buffer,water and prepGEM
  4. Incubate in a thermal cycler
  5. Centrifuge for 2 minutes at max speed and transfer the supernatant to a fresh tube.
  6. Do PCR
67
Q

What is the use of prepgen extraction kit

A
  1. Resolves inconsistent amplification
68
Q

What is the use of maxwell dna extraction

A
  1. Automated dna extraction
  2. Can use blood samples and tissue samples
69
Q

What is direct amplification

A
  • Method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification or quantification.
  • blood, baccalaureate swabs and tissue samples are used.
70
Q

Capillary electrophoresis

A
  1. Fragments are separated by size
  2. Before reaching the anode, fragments pass through the laser.
  3. Laser beam causes the fluorescent dyes to fluoresce at different emission wavelengths
  4. CCD camera captures the fluorescence intensities.
  5. Theses are digitalised and colour coded
  6. Displayed as peaks on the electropherogram.
71
Q

Why are controls used during PCR and capillary electrophoresis.

A

+ve control = gives an indication of wether the PCR conditions and reagents are working.
-ve control= detects contamination

72
Q

Characterise verifier kit profile

A
  1. Six-colour fluorescent
  2. 75-465bp
  3. 23 autosomal str loci
  4. Y indel-polymorphic
  5. Amelogenin
  6. 25-loci multiplex
  7. Proffered for kinships
  8. Currently used routinely for parentage
73
Q

Characterise powerplex 18D kit profile

A
  1. Five-colour fluorescence.
  2. 17 autosomal STR and Amel
  3. 18 loci-multiplex
74
Q

Potential problems with analysis of DNA Profiles

A
  1. Motherless tests
  2. Allelic drop-out /null alleles
  3. Off-ladder alleles
  4. Contamination
  5. Identical twins
  6. Pull ups
75
Q

Explain what a Allelic dropout is.

A

When one or both alleles of a heterozygote fail to amplify resulting in the peak not detected during electrophoresis.

76
Q

Explain what a null allele is.

A

Primer in the PCR reaction fails to hybridise to the template DNA

77
Q

Explain what off ladder alleles are

A
  1. Alleles that are not exact multiples of the basic repeat motif.
  2. These alleles are not labelled by the allelic ladder.
78
Q

What are the two outcomes for paternity testing ?

A
  1. Excluded : the obligate paternal alleles in the child all have corresponding levels in the alleged father.
  2. Not excluded: the obligate paternal alleles in the child do not have corresponding alleles in the alleged father.
79
Q

Define Paternity index (PI)

A
  1. Compares the likelihood that a genetic mark (allele) that the alleged father passed to the child to the probability that a randomly selected unrelated man of similar tonic background could pass the allele to the child.
  2. Represented by formula PI =X/Y
    X= represents the chance that the alleged father could transmit the obligate allele
    Y= represents the chance that another man of the same race cold have transmitted the allele.
80
Q

What is the combined paternity index

A
  1. CPI is an odds ratio that indicates how many times more likely it is that the alleged father is the biological father than a randomly selected unrelated man of similar ethnic background.
  2. CPI= PI of STR1 x PI x STR2 x PI xSTR3 ……..
81
Q

How to calculate probability of paternity (p)

A
  1. CPI/(1+CPI) x 100
  2. CPI>100 = inclusion
  3. P>99%=supports the relationship
82
Q

What is kinship testing ?

A
  1. Kinship testing is a process if determining reltetedness between individuals where first degree relatives such as parents are not available for testing.
  2. As the genetic distance between individual increases, the probability of determining relatedness decease.
83
Q

What are the additional testing done if the first line autosomal testing comes out inconclusive .

A
  1. Mitochondrial
  2. X-chromosome
  3. Y-chromosome
84
Q

In kingship testing, what are the three results that can be expected for autosome testing .

A
  1. Not excluded: CPI>100
  2. Inconclusive results: CPI: 0,1-99
  3. Excluded: CPI<0,1
85
Q

Explain X-chromosome testing.

A
  1. Used for female siblings allegedly sharing same father or child and alleged paternal grandmother.
  2. Argus X-12 Qs kit which contains 12 X-chromosomal short tandem repeat (STR) loci and AMEL.
86
Q

Explain mtDNA testing

A
  1. Used for half siblings who may share a mother and child and maternal bloodline.
  2. Sanger sequencing of HVRI and HVRII. If they are different =unlikely related. If they are the same = related.
87
Q

Explain Y-chromosome testing

A
  1. Used for half male siblings with the same alleged father and child and alleged paternal male family members.
  2. AmpFLSTR Yfiler PCR Amplification kit. It amplifies 17 Y-STR loci in a single PCR.

GH Coarsework 2023 (13 decks)

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