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Flashcards in techniques Deck (36)
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1
Q

PCR

A
amplifies DNA using thermocycle
1) 96
2)65
3) 72
x 30 times
- run across agarose gel to separate stands
2
Q

after PCR

A

gel electrophoresis. This technique separates fragments by charge, size (molecular weight) and shape

3
Q

viral DNA will never be longer than

A

300bp

4
Q

qPCR

A

amplifies and quantifies

- can be used to see if certain genes ar being over expressed or bacterial, fungal or viral load

5
Q

which fluorescent dye is used in qPCR

A

SYBR

6
Q

SYBR can only bind to

A

double stranded DNA

7
Q

qPCR proces

A

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

8
Q

Ct

A

cycle threshold- the number of cycles required for fluorescence to pass the threshold

9
Q

GWAS

A

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
10
Q

what does GWAS look for

A

SNPs

11
Q

SNPs

A

insertions/ deletions- which could cause frameshifts/ ORF - which could causes misfolding of proteins and therefore disease

12
Q

GWAS is useful in

A

finding genetic variations which contribute to common, complex diseases e.g. diabetes, asthma, cancer

13
Q

GWAS is an example of

A

hypothesis free testing

14
Q

if we know which SNPs causes diseases

A

we can design specific primers and PCR to scan for them

15
Q

how could GWAS be used as a case-control experiment

A
  • 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
16
Q

how could GWAS be used in a cohort study

A

show genotype to phenotype correlation

- how far disease are environmental and how far they are genetic

17
Q

sanger method

A

used to sequence DNA

18
Q

outline sanger

A

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

19
Q

why is NGS superior to stager sequencing

A
  • good for generating big data
  • quicker
  • can determine the DNa sequence of a large number of diff DNA molecules
20
Q

How to NGS computers reassemble the genome

A

looking for overlapping sequences

21
Q

NGS basics

A

gene sequencing, cDNA sequencing (viral)

22
Q

NGS can also be used

A

look for point mutations

23
Q

NGS methods

A

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

24
Q

why does NGS require deep sequencing

A

-multiple readings to ensure sequences are true reflections

25
Q

CRISPR can be used to

A

cure inherited disease, kill cancer and viral cells as well as genetically modifying plants and animal

26
Q

basic CRISPR process

A

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

27
Q

how is CRISPR introduced to cells

A
  • added into plasmids and then injected into cells using virus’
28
Q

CRISPR plasmid must have

A

sgRNA with Cas9 binding sequence down stream of promotor (PAM) NGG
- also need Cas9 cDNA

29
Q

were can CRISPR bind

A

NGG

- also think of the complementary stranded of DNA so NCC too

30
Q

CRISPR can also be used to kill

A

virus’ inside cells e.g. if the DNA of virus has been deduced

31
Q

how can CRISPR cause deletion of bad genes

A

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

32
Q

homologous recombination can be used to

A

introduce a good form of a gene into a cell

33
Q

RNA sequencing

A

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

34
Q

RNA sequencing leaves you with

A

a list of ones which are unregulated or down regulated in hypoxic cells

35
Q

process of RNA sequencing

A

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

36
Q

RNA sequencing simple

A
  • purify mRNA
  • add RT , mRNA- cDNA
  • sequence in NGS