Genetics 6 - Molecular Genetics and Sequencing Flashcards
molecular genetics definition
refers to all diagnostic methods that examine changes of genetic info in extracted DNA or RNA
2 main approaches for diagnosis
- nucleic acid hybridisation - based on complementarity (of nucleic acid sequences on 2 pieces of DNA)
- nucleic acid amplification - making many DNA copies (ability. of polymerase chain rxn to amplify up DNA)
4 categories of test and what they are used for
mutation scanning - is there any mutation present
mutation screening - is a known mutation present
genomic quantification - is there extra/missing DNA
sequencing - what is the exact DNA/RNA sequence
GOLD STANDARD
hybridisation process
sense (coding) and antisense (template) strands are hybridised to each other in a dsDNA molecule
when heated to 95°C the strands denature (break apart)
complimentary sequences can then hybridise to each strand
amplification (PCR) process following hybridisation
primer sequences can hybridise (anneal) to each strand at 55-60°C
in the presence of dNTPs and DNA polymerase the complimentary DNA strand is extended in each direction (72°C)
⇒ 2 amplicons have the same sequence (copies)
amplification ⇒
each cycle doubles the number of amplicons
exponential amplification of the target DNA sequence
2 mutation scanning methods
what do they examine
based on….
SSCP - single-strand conformation polymorphism
DGGE - denaturing gradient gel electrophoresis
examine a gene for all possible mutations (including unknown) - is there any mutation present?
based on analysis of physical or chemical characteristics of PCR product (hybridisation and amplification)
abnormalities must be confirmed by sequencing
SSCP and DGGE - how do they work (don’t need to know detail)
mutation screening methods - for a known mutation
is a known mutation present
PCR based screening for known specific mutations
indicated in diseases with few discrete but frequent mutations
need to know sequence you are looking for to design specific primers
can check presence/absence and size of specific sequence of 50bp to 5kb (RFLP)
METHODS:
PCR
OLA
Southern blotting/RFLP
PCR - how does it work
detects specific KNOWN mutations
can check presence/absence and size of PCR product
allele-specific PCR and the oligonucleotide ligation assay
PCR will not leave product if 3’ most primer is not correctly base paired
southern blotting/RFLP
detects large scale changes in DNA fragments
e.g. long repeat extensions in triplet repeat disorders
molecular analysis for Fragile X Syndrome and Huntington Disease
molecular analysis for Fragile X Syndrome and Huntington Disease
Southern Blotting/RFLP
genomic quantification methods - extra/missing DNA
looks at
detects
use in cases of…
limitations
methods
is there extra/missing DNA
can look at whole genome on 1 microarray
detect duplications, heterozygous deletions, copy number variability, unbalanced structural abnormality at resolution of 30kb
indicated in cases of multiple malformations or unknown dysmorphism
limitations =can’t detect balanced rearrangements (inversions and translocations), small deletions or duplications, point mutations, expanded trinucleotide repeats
⇒ not suitable for most MONOGENIC disorders
METHODS:
DNA microarrays
SNP arrays (SNP chips)
what methods are not suitable for most monogenic disorders
DNA microarrays
SNP arrays (SNP chips)
DNA microarray analysis
SNP array - SNP chip
Uses allele specific oligonucleotides
Only DNA of test subject used
Detects duplications and deletions with greater resolution than microarray
Homo and heterozygosity measured
Di-deoxy (Sanger) sequencing
next generation sequencing (NGS)
ultra high throughput
high accuracy and flexibility
dramatically cheaper than Sanger
large range of “omics” applications - metagenomics, proteomics, epigenomics
things to remember
summary of thalassaemias
