techniques Flashcards
PCR
amplifies DNA using thermocycle 1) 96 2)65 3) 72 x 30 times - run across agarose gel to separate stands
after PCR
gel electrophoresis. This technique separates fragments by charge, size (molecular weight) and shape
viral DNA will never be longer than
300bp
qPCR
amplifies and quantifies
- can be used to see if certain genes ar being over expressed or bacterial, fungal or viral load
which fluorescent dye is used in qPCR
SYBR
SYBR can only bind to
double stranded DNA
qPCR proces
1) same as PCR, although if using mRNA (virus), then RT will need to be used to produce cDNA
2) compare control gene in normal + e.g. cancerous cell (NADPH) by using Ct
3) then compare gene of interest in normal and cancerous cell by using Ct
Ct
cycle threshold- the number of cycles required for fluorescence to pass the threshold
GWAS
genome wide association study
- a study being conducted to help inform us which genes are involved in certain diseases
e. g. helping to determine genetic factors of diseases
what does GWAS look for
SNPs
SNPs
insertions/ deletions- which could cause frameshifts/ ORF - which could causes misfolding of proteins and therefore disease
GWAS is useful in
finding genetic variations which contribute to common, complex diseases e.g. diabetes, asthma, cancer
GWAS is an example of
hypothesis free testing
if we know which SNPs causes diseases
we can design specific primers and PCR to scan for them
how could GWAS be used as a case-control experiment
- take a group of diseased people and a group of healthy people
- see if there is an over representation of SNPs in disease patients
e. g. could be used to help drug targeting
how could GWAS be used in a cohort study
show genotype to phenotype correlation
- how far disease are environmental and how far they are genetic
sanger method
used to sequence DNA
outline sanger
1) DNA sample denatured, leaving single stranded DNA
2) high fidelity DNA polymerase is added along with nucleotides and fluorescently labelled stop nucleotides
3) when a fluorescent A, T, G ,C nucleotides binds, it prevents DNA polymerase from synthesising any more DNA
4) the fluorescent emission of diff nucleotides are detected by lasers as the DNA flows through gel electrophoresis via capillary action
5) allowing order to be determined
why is NGS superior to stager sequencing
- good for generating big data
- quicker
- can determine the DNa sequence of a large number of diff DNA molecules
How to NGS computers reassemble the genome
looking for overlapping sequences
NGS basics
gene sequencing, cDNA sequencing (viral)
NGS can also be used
look for point mutations
NGS methods
1) DNA extraction and fragmentation
2) machine separates each fragment
3) fragments then amplified using PCR
4) coloured nucleotides (one at a time e.g. only C first) added allowing order to be decked
5) process repeated until nucleotides sequenced
why does NGS require deep sequencing
-multiple readings to ensure sequences are true reflections
CRISPR can be used to
cure inherited disease, kill cancer and viral cells as well as genetically modifying plants and animal
basic CRISPR process
1) sgRNA is introduced and has an identical sequence to the target gene
2) sgRNA complementary base pairs with target gene and its secondary structure is able to recruit Cas9
3) Cas9 then cleaves the dsDNA, leaving a straight break
how is CRISPR introduced to cells
- added into plasmids and then injected into cells using virus’
CRISPR plasmid must have
sgRNA with Cas9 binding sequence down stream of promotor (PAM) NGG
- also need Cas9 cDNA
were can CRISPR bind
NGG
- also think of the complementary stranded of DNA so NCC too
CRISPR can also be used to kill
virus’ inside cells e.g. if the DNA of virus has been deduced
how can CRISPR cause deletion of bad genes
Homologous end joining (NHEJ) once the Cas9 has cleaved the double stranded DNA, homologous end going occurs
-often causes deletion/ insertion–> disrupting the reading frame and preventing production of the protein
homologous recombination can be used to
introduce a good form of a gene into a cell
RNA sequencing
quantifies how much mRNA of each gene you have in a cell e.g. if you wanted to see difference in expression of genes in a normoxic vs hypoxic cell
RNA sequencing leaves you with
a list of ones which are unregulated or down regulated in hypoxic cells
process of RNA sequencing
1) purify mRNA form both cells
2) revise transcriptase mRNA- cDNA
3) cDNA copy of the mRNA is added to NGS machine
4) gives quantification of how much gene you had in your starting sample
5) looks at how gene expression is different in normoxic vs hypoxic cells
RNA sequencing simple
- purify mRNA
- add RT , mRNA- cDNA
- sequence in NGS
