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
