DNA variation and markers Flashcards
(42 cards)
DNA variants
SNP
deletion
insertion
short tandem repeat
methods used in genetic testing
restriction fragment length polymorphism (RFLP)
Allele specific oligonucleotides (ASO)
Allele specific PCR (ARMS)
sequencing (NGS)
up to 3billion bp targeted (individual genes) exome, whole genome short processing time (24hrs-3 weeks) depending on how large the target is cost - thousands
Genotyping
- Up to 5 million bp (usually 5-600k)
- Targeted (individual to few SNPs)
- Small scale (dozens to hundreds SNPs) • Medium scale (thousands of markers)
- Large-scale (500k – 5 million)
- Processing time usually a few days
- Cost varies, most around 50 AUD
sequencing vs genotyping
complete information vs partial all variants vs known variants expensive vs cheap slower vs faster diagnosis vs risk, prevention
SNP
one base pair difference
RFLP
difference in DNA sequences that can be detected by fragments of different lengths after restriction enzyme cut the surrounding sequence
detect RFLP via PCR
PCR primers are added to DNA sequence for amplification, then spliced by a restriction enzyme and run on agarose gel
ASO
probe hybridised onto DNA strand. there is a strand identical but with a single base pair mismatch which prevents the probe from hybridising to it. when the temp and run thru machine is raised the difference in normal and against homozygote and heterozygote
for wildtype probe
normal = colour
mutation = white
for mutant probe
normal = white
mutation = colour
ASO in sickle cell anaemia
when a probe is used for the normal allele
- colour will show with homo and het wild-type - due to binding = colour
- white for mutation - mismatch no binding
when a probe is used for the mutatant allele
- colour will show with homo and het mutant - due to binding = colour
- white for wildtype (normal) - mismatch no binding
ASO in pre-implantation genetic diagnosis (PGD)
DNA from isolated cell
amplified by PCR for the b-globin gene
one hybridised probe for normal allele and mutant allele
ASO detect small inDels
can detect small deletions
eg. CF
one for normal sequence in region CF sequence
one for mutant CF sequence around deletion
if there is white (no binding) to normal probe and colour (binding) to deletion probe = CF
ASO scanning mutations throughout specific gene
using multiple sets of different probes for different SNP mutations you can detect mutations
ARMS
single DNA as a probe
- outer primers - amplify all alleles
- inner primers - specific for wild-type alleles
e. g. G (WT) or T (mutant) - primer for G will bind to G if it is at a specific position and give a PCR product (only if they are G/G homo or G/T het)
primer for T will bind to T if it is at a specific position and give a PCR product (only if they are T/T homo or G/T het)
put all 4 primers, run PCR look at band sizes and determine if homo WT or mutant or het
Why are SNPs good for mapping
very common
- SNP occur once every ~300 bp
- in 3,000,000kb there are 10m SNPs
limitations
only have 2 alleles
satellite loci have many alleles
NGS steps
- DNA fragmentation
- library preparation (primers, adapters)
- template amplification (PCR, 3rd generation sequencing) 4. Sequencing (by synthesis, ligation etc.)
- Assembly
NGS: assembling contigs
genomic DNA is cut in overlapping segments by digestion with restrictive enzymes to create continuous fragments (“contigs”)
overlapping sequenced fragments aligned using machine to assemble entire chromosome
fragments are aligned based on identical sequences
NGS applications
genome sequencing
metagenomics, microbiota studies (identifying the microbiota of varies organs on the body)
diagnostic RNA-seq expression profiling (looking at RNA for diseases)
mutation/SNV detection
satellite loci
Short DNA sequences that occur in a variable number of tandem repeats in genome.
Number of repeats varies between individuals and between the two chromosomes in a given individual.
micro-satellite
2-10 bp (short tandem repeat)
mini-satellite
10-100 bp (variable number of tandem repeats, VNTR)
STR and VNTR detected by gel electrophoresis
i) Southern hybridisation - using the repeat sequence as a probe (VNTRs) or
ii) PCR - using sequences on each side of the repeat as primers (STRs)
are satellites and ASO complete dominant
no - they are co-dominant thus the heterozygote is recognisable
satellites for inheritance
PCR primers select for micro-satellite repeats
