techniques

Question 1

PCR

Answer 1

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

Question 2

after PCR

Answer 2

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

Question 3

viral DNA will never be longer than

Answer 3

300bp

Question 4

qPCR

Answer 4

amplifies and quantifies

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

Question 5

which fluorescent dye is used in qPCR

Answer 5

SYBR

Question 6

SYBR can only bind to

Answer 6

double stranded DNA

Question 7

qPCR proces

Answer 7

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

Question 8

Ct

Answer 8

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

Question 9

GWAS

Answer 9

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

Question 10

what does GWAS look for

Answer 10

SNPs

Question 11

SNPs

Answer 11

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

Question 12

GWAS is useful in

Answer 12

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

Question 13

GWAS is an example of

Answer 13

hypothesis free testing

Question 14

if we know which SNPs causes diseases

Answer 14

we can design specific primers and PCR to scan for them

Question 15

how could GWAS be used as a case-control experiment

Answer 15

• 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

Question 16

how could GWAS be used in a cohort study

Answer 16

show genotype to phenotype correlation

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

Question 17

sanger method

Answer 17

used to sequence DNA

Question 18

outline sanger

Answer 18

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

Question 19

why is NGS superior to stager sequencing

Answer 19

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

Question 20

How to NGS computers reassemble the genome

Answer 20

looking for overlapping sequences

Question 21

NGS basics

Answer 21

gene sequencing, cDNA sequencing (viral)

Question 22

NGS can also be used

Answer 22

look for point mutations

Question 23

NGS methods

Answer 23

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

Question 24

why does NGS require deep sequencing

Answer 24

-multiple readings to ensure sequences are true reflections

Question 25

CRISPR can be used to

Answer 25

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

Question 26

basic CRISPR process

Answer 26

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

Question 27

how is CRISPR introduced to cells

Answer 27

• added into plasmids and then injected into cells using virus’

Question 28

CRISPR plasmid must have

Answer 28

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

Question 29

were can CRISPR bind

Answer 29

NGG

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

Question 30

CRISPR can also be used to kill

Answer 30

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

Question 31

how can CRISPR cause deletion of bad genes

Answer 31

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

Question 32

homologous recombination can be used to

Answer 32

introduce a good form of a gene into a cell

Question 33

RNA sequencing

Answer 33

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

Question 34

RNA sequencing leaves you with

Answer 34

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

Question 35

process of RNA sequencing

Answer 35

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

Question 36

RNA sequencing simple

Answer 36

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