Genetic testing Flashcards
two main types of genetic testing
molecular testing
cytogenetics
molecular testing
sequencing of specific genes or genotyping specific small mutations based on phenotype
what does molecular testing use
PCR and sequencing
types of molecular testing
whole genome sequencing
whole exome sequencing
whole genome sequencing
the process of determining the complete DNA sequence of an individual at one time
process of WGS
1) obtain DNA sample
2) purify DNA and break into short fragments
3) sequence many times over
why sequence the whole genome
to find the cause of an unexplained disease that appears to be genetic
Whole exome sequencing
sequencing of all exomes in the genome
exomes
expressed part of the genome and some regulatory sequences
how is the exome filtered out of the exome
DNA gets fragmented and passed through a filter which traps the coding sequences (complimentary binds with exome ssDNA)
the exome genome is compared to
a start human reference genome sequences
what can be found in WES
presence of known SNP polymorphism
rare differences
unique differences
INDELS
why sequence an exome
cheaper than sequencing the whole genome
- get almost as much useful info as WGS
What is PCR sued to do
amplify specific segments of DNA
why is PCR important
amplification allows detection of pathogenic virus’ and bacteria, as well as identification of individuals e.g. in crime
what ingredients are needed for PCR
1) primers
2) dNTPs (free nucleotides)
3) Taq polymerase
4) DNA template to be copied
5) buffer
what is used in PCR
a thermocycler
process of PCR
1) denaturing (90-95)- separate double strands
2) annealing (50-56)- temp lowered to enable primers to bind
3) extension (72)- temp raised and new strands synthesised by polymerase
–> cycle repeated- DNA doubles everytime
how is whole genome sequence glad whole exome sequencing carried out
using NGS
what was first generation sequencing
Sanger
outline the sanger method
- DNA to be sequenced is amplified (PCR)
- DNA is then denatured using heat– ssDNA
- DNA split into template and complimentary strand
- primer for specific region of DNA is then annealed to the template strand to allow addition of nucleotides
- 4 reaction mixtures are set up
- in each mixture the template strand of DNA, DNA polymerase and free nucleotides (dNTP are added)
- modified ddNTPs (tagged with fluorescence) are then added to the mixture (one type of fluorescent A, T, C ,G added to each well)
- ddNTPs cause termination of polymerisation due to lacking a hydroxyl group
- PCR occurs- by the end each mixture will yield PCR products of differing lengths due to replication being randomly terminated
- DNA then separated on the basis of six on a gel electrophoresis page
- can detect order when an x ray is used due to fluorescent bands that appear
next generation sequences method is also called
second generation
why is NGS better than first generation (sanger)
- more accurate
- quicker
- reduced manpower
- reduced cost
name a type of NGS
e.g. pyrosequencing
name 3 cytogenetic testing methods
- karyotyping
- FISH
- microarrays
karyotyping is a
cytogenetic test
karyotyping is the process of
ordering and pairing all chromosomes of an organism
chromosome 1 is the
largest chromosome
chromosome 22
is the smallest
karyotyping is a test to…
identity the size, shape and number of chromosomes in a sample of body cells
how many chromosomes in body cell
46
autosomes
Chr1-22
–> 44 autosomes
karyotyping can be used to detect
- abnormal numbers of chromosomes
- abnormal size e.g. CNVs
name two karyotyping methods
- light microscope
- G-banded karyotypes
light microscope: karyotyping
can detected abnormal number of chromosomes
can detected abnormal number of chromosomes
aneuploidy/ polyploidy
G-BANDED KARYOTYPE
shows structural problems with chromosome
what stain is used in G-banded karyotype
Giesma stain
- stains condensed chromosomes
- causes dark bands
Gismo stains which part of the chromosomes
AT rich regions– can show homologous chromosomes e.g. will have bands in the same place
microarray is another type of
cytogenetic test
microarrays are a
tool used to detect the expression of thousands of genes at the same time
microarray process
1) mRNA extracted from reference (healthy) and experimental sample (patient)
2) mRNA is reverse transcribed to cDNA
3) reference cDNA is tagged green
4) experimental cDNA is tagged red
5) both cDNA samples are combined and added to a microarray chip
6) cDNA binds to its complimentary base pair which is attached to the chip
7) chip is rinsed
8) chip put through a laser scanner (which activates fluorescent dye)
9) indicates which evens are being expressed (if they ar being expressed differently)
if yellow
both genomes express the same gene
name two types of microarray
SNP-array
Array- CGH
SNP- array detect
single nucleotide polymorphisms (most common type of mutation)
Array-CGH
comparative genomic hybridisation
Array-CGH analyses
CNVs
CNV
copy number variants
FISH is a type of
cytogenetic test
FISH stands for
fluorescence in situ hybridisation
FISh was developed to
detect and locate presence or absence of specific DNA sequence on the chromosome
aim of FISH
to detect abnormal chromosomes- caused by the binding of fluorescent probe that is complimentary to a specific region on a chromosome
- shows if chromosome location is mutated or not
what are standard diagnosis tests
- microarrays
- molecular testing for specific conditions
targeted next regeneration sequencing
gene panels based on phenotypes
genome wide sequencing using NGS
- whole exome sequencing
- whole genome sequencing
how many protein coding genes
20,000
what else can be sequences
the clinical exome
the clinical exome
includes are 4,000 known disease genes
