Techniques 1 Flashcards
What are numerical chromosome abnormalities
- Aneuploidy
- Polyploidy
What are structural chromosome abnormalities
- Deletions
- Duplications
- Translocation
- Inversion
- Insertion
Characterise polyploidy
- More than 2 haploid chromosome sets
- Example: triploidy = 3 haploid chromosome sets =69 chromosomes
Characterise Aneuploidy
- Gain or loss of one chromosome
- Example: trisomy = 46+1=47 chromosomes
Explain what happens during Non-disjunction in meiosis 1
- Two homologous chromosome fail to separate
- Results in trisomic or nullisomic embryos
Explain what happens during non-dysfunction in meiosis 2
- Two sister chromatids don’t separate
- Results in trisomic or monosomic embryos
Mechanisms resulting in polyploidy
- Digyny
- Diandry
Define digyny
Diploid ovum is fertilised by a haploid spermatozoa
Define diandry
- Fertilisation of one oocyte by two spermatozoa.
- Four times more frequent that digyny.
Define mosaicism
- Occurs when there are two or more different cell lines present.
Define chimerism
- Two or more cell lines present originating from two or more zygotes
Name of the trisomy 13
Palau syndrome
Clinical features of Down syndrome
- Characteristic dystrophic features
- Intellectual delay
- Global development delay
- Hypotonia
- Congenital heart defects
- Hearing loss
- Vision problems
- Hypothyroidism
Clinical feature of Palau syndrome
- Severe intellectual disability
- Severe global development delay
- Congenital abnormalities of various organ systems
- Holoprosencephaly
- Polydactyly
- Cleft lip and/or palate
- Failure to thrive
- Poor prognosis
Name for trisomy 18
Edwards syndrome
Clinical features for Edward syndrome
- Severe intellectual disability
- Severe global developmental delay
- Microcephalic
- Clenched fists with overlapping digits
- Rocker bottom feet
- Congenital malformations of various organ system
- Poor prognosis
Turner syndrome is caused by
Missing X chromosome - 45,X
Clinical features of Turner syndrome
- Short stature
- Dysmorphic puberty
- Delayed puberty
- Infertility
- Lymphoedema
- Kidney abnormalities
- Cardiac abnormalities
Cause of Klinefelter syndrome
Males with an extra X chromosome - 47 XXY
Clinical feature of Klinefelter syndrome
- Tall stature
- Reduced muscle tone
- Small testes
- Delayed pubertal development
- Decreased facial and body hair
- Gynaecomastia
- Speech and language delays
- Behavioural problems
Characterise deletions
- Part of the chromosome is missing or deleted
- Some genetic material is missing can cause syndrome features, this depends on the genes involved in deletion.
- Can be sporadic or inherited
Different types of deletions.
- Interstitial deletion ( in the middle)
- Terminal deletion ( at the ends )
Example of diseases caused by deletions
- Wolf-hirschhorn syndrome -4p deletion
- Cri du chat syndrome - 5p deletion
Characterise Duplication
- Part of the chromosome is duplicated
- Results in extra genetic material, severity depends on the genes involved.
- Often less severe than deletions
- Can be. Sporadic or inherited
Diseases caused by duplications
22q duplication syndrome -22q11 duplication
Explain translocation ; reciprocal
- Exchange of maternal between chromosomes
What are balanced translocations
- Non loss or gain or genetic material.
- Individuals are usually phenotypically normal
What are unbalanced translocations
- Gain or loss of genetic material -severity depends on size
- Can be inherited from a balanced parent -recurrence is complicated
Explain what robertsonian translocation
- Exchange between two acrocentric chromosome to form mine large metacentric chromosome
Describe inversion
- Portion of a chronometer broke off, inverted and reattached.
- Can be Paracentric = on one arm
- Or pericentric = between the two chromosome
- If isolated the individual is often phenotypically normal
Describe insertions
- Proportion of a chromosome broke off and is inserted elsewhere.
- Interchromosomal = 2 different chromosomes
- intrachromosomal = within a single chromosome
- Direct insertion or inverted insertion
Characterise Ring chromosome
- Portion of a chromosome broke off and formed a ring
- Can happen with or without loss of genetic material
3, very rare - Often severe
- Usually sporadic
Characterise isochromosome
- Formed by a mirror image copy of a chromosome segment
- Results in a gain and loss of genetic material
Diseases caused by isochromosome
- Isochromosome 12 = pallister killan syndrome
- Turner syndrome =isochromosome Xq
Clinical indications for congenital cytogenetic prenatal testing
- Abnormal ultrasound findings
- Abnormal maternal serum screening
- Familial or parental chromosome abnormality
Clinical indications for congenital cytogenetic postnatal
- Newborn/childhood growth and development problems.
- Adolescent/adult sexual development ad fertility. I.e amenhorrhea, history of miscarriages
Clinical indications for molecular cytogenetic test FISH
- Micro-deletions/ duplication syndromes
- Confirm rearrangements suspected on a karyotype.
3.Advance maternal age - Birth defects or neural tube defects on ultrasound
Clinical indications for molecular cytogenetic test microarray
- Unexplained multiple congenital abnormalities, dysmorphism, gross development delays, behavioural problems.
Clinical indications for molecular cytogenetic test QF-PCR
- Rapid screening for common Aneuploidy chromosome.
- Advanced maternal age >35 years
- Sex determination (XX/XY)
Function of L-glutamine (amino acid) in culturing
- Supplementary energy source for rapidly dividing cells.
- Serve as source of nitrogen for synthesis of proteins and nucleic acids
Function of foetal bovine serum (FBS) in culturing
- Provide nutrients for cell growth
Function of Antibiotics in culturing
Prevent microbial growth
Function of phytohemagglutin (PHA)
- Mitogen which binds to the T-cells
- Thus stimulates cell division
- Resulting in mitotic cells
Function of colcemid
- Arrests cells at prophase or early metaphase stage, while the chromosome are condensed and still intact.
How does colcemid work.
- Binds to the spindle apparatus
- Preventing the formation of spindle fibres
- Resulting in spindle fibers not being able to attach to the centromeres
- Thus sister chromatids cannot be pulled into the opposite poles of the cells.
- Also stops the activity of formed spindle fibers.
What is hypotonic treatment
- Process of increasing the cell volume
- In order to increase the cell surface area
- So that chromosome may evenly spread out
Function of KCl/ sodium citrate
- Creates a concentration gradient across the cytoplasmic membrane.
- Thus allowing water to move from high concentration
- Resulting in the cell absorbing H2O and swelling
- Thus increasing the cell volume so that chromosome have sufficient space to spread out.
Precautions and considerations of hypotonic treatment.
- Type of salt used can impact with width and sometimes the length of chromatids produced.
- Extended exposure to the hypotonic solution can cause the membrane to burst and allow some chromosome to escape.
Explain the process of fixation.
- Lysine of erythrocytes and proteins.
- Preventing cells from further swelling
- Preserving the morphology of metaphase cells
- Hardening the cell membrane and chromatids thus preparing then for banding.
Fixation of chromosomes in karyotyping is achieved by using?
- 3:1 methanol-acetic acid
- Kept in cold temperature
Disadvantages of chromosomal analysis
- Requires fresh samples
- Prolonged culturing
- Prone to culture failure resulting in suboptimal or unsuccessful results.
- Microdeletions/duplications or cryptic rearrangements cannot be detected.
- Abnormalities caused by molecular genetic mutations cannot be detected.
Making a slide depends on what variables.
- Height
- Air flow
- Slide quality
- Wet vs dry slide
- Angle of the slide
- Humidity / temperature
What is aging during slide making
- Leads to cells becoming dehydrated.
- Thus allows metaphase cells to withstand further treatment during banding.
What is differential banding and give examples
- Produces thin, alternating bands along the length of the entire chromosome.
- Examples: G-banding , R-banding, Q-banding
What is selective banding and give examples
- Stains only a specific band or region.
- C- Banding and NOR staining
Uses of G-banding
- Detection f both structure and numerical chromosome abnormalities
- Most commonly used banding method
Uses of R-banding
- Detection of chromosomal rearrangements that involve the telomeres
Uses of Q-banding
- Identifying the hetechromatin regions of certain chromosomes such as Y-chromosome and acrocentric chromosomes.
- Help to identify structural rearrangements involving Y-chromosome
Uses of c-banding
- Identifying the number of centromeres present in a chromosomal rearrangement.
- Visualisation of constitutive heterochromatin at the centromeric regions of chromosome 1,9,16 and long arm of Y-chromosome.
Use of Nor -staining
- Identify the stalk regions of acrocentric chromosomes especially when involved in translocations or in a marker chromosome.
- Confirm or exclude the presence of big satellites of acrocentric chromosomes
Why would you do parentage testing.
- Asked for Magistrate/high court.
- Asked for by home affairs
- Private
- disease related
- Forensics
- Asked for by hospital
Types of test done by DPL
- Trio : mother, child and alleged father
- Duo; father + child or mother +child
- Kinship: testing of a child and relative
Why short tandem repeats area used in DPL.
- Repeat sequences of non-coding DNA
- Polymorphic
- Number of repeats is inherited
- Therefore, unrelated individuals unlikely to have the same number.
What do DPL use for DNA extraction
Roche extraction kit
Sample processing in DPL.
- Receiving
- DNA extraction
- Amplification
- Capillary electrophoresis
- Analysis of results
- Reporting
Explain PREGEM Extraction steps.
- 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.
- 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.
- Add buffer,water and prepGEM
- Incubate in a thermal cycler
- Centrifuge for 2 minutes at max speed and transfer the supernatant to a fresh tube.
- Do PCR
What is the use of prepgen extraction kit
- Resolves inconsistent amplification
What is the use of maxwell dna extraction
- Automated dna extraction
- Can use blood samples and tissue samples
What is direct amplification
- 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.
Capillary electrophoresis
- Fragments are separated by size
- Before reaching the anode, fragments pass through the laser.
- Laser beam causes the fluorescent dyes to fluoresce at different emission wavelengths
- CCD camera captures the fluorescence intensities.
- Theses are digitalised and colour coded
- Displayed as peaks on the electropherogram.
Why are controls used during PCR and capillary electrophoresis.
+ve control = gives an indication of wether the PCR conditions and reagents are working.
-ve control= detects contamination
Characterise verifier kit profile
- Six-colour fluorescent
- 75-465bp
- 23 autosomal str loci
- Y indel-polymorphic
- Amelogenin
- 25-loci multiplex
- Proffered for kinships
- Currently used routinely for parentage
Characterise powerplex 18D kit profile
- Five-colour fluorescence.
- 17 autosomal STR and Amel
- 18 loci-multiplex
Potential problems with analysis of DNA Profiles
- Motherless tests
- Allelic drop-out /null alleles
- Off-ladder alleles
- Contamination
- Identical twins
- Pull ups
Explain what a Allelic dropout is.
When one or both alleles of a heterozygote fail to amplify resulting in the peak not detected during electrophoresis.
Explain what a null allele is.
Primer in the PCR reaction fails to hybridise to the template DNA
Explain what off ladder alleles are
- Alleles that are not exact multiples of the basic repeat motif.
- These alleles are not labelled by the allelic ladder.
What are the two outcomes for paternity testing ?
- Excluded : the obligate paternal alleles in the child all have corresponding levels in the alleged father.
- Not excluded: the obligate paternal alleles in the child do not have corresponding alleles in the alleged father.
Define Paternity index (PI)
- 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.
- 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.
What is the combined paternity index
- 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.
- CPI= PI of STR1 x PI x STR2 x PI xSTR3 ……..
How to calculate probability of paternity (p)
- CPI/(1+CPI) x 100
- CPI>100 = inclusion
- P>99%=supports the relationship
What is kinship testing ?
- Kinship testing is a process if determining reltetedness between individuals where first degree relatives such as parents are not available for testing.
- As the genetic distance between individual increases, the probability of determining relatedness decease.
What are the additional testing done if the first line autosomal testing comes out inconclusive .
- Mitochondrial
- X-chromosome
- Y-chromosome
In kingship testing, what are the three results that can be expected for autosome testing .
- Not excluded: CPI>100
- Inconclusive results: CPI: 0,1-99
- Excluded: CPI<0,1
Explain X-chromosome testing.
- Used for female siblings allegedly sharing same father or child and alleged paternal grandmother.
- Argus X-12 Qs kit which contains 12 X-chromosomal short tandem repeat (STR) loci and AMEL.
Explain mtDNA testing
- Used for half siblings who may share a mother and child and maternal bloodline.
- Sanger sequencing of HVRI and HVRII. If they are different =unlikely related. If they are the same = related.
Explain Y-chromosome testing
- Used for half male siblings with the same alleged father and child and alleged paternal male family members.
- AmpFLSTR Yfiler PCR Amplification kit. It amplifies 17 Y-STR loci in a single PCR.
