Week 8

Question 1

what methods are used for nucleic acid detection

Answer 1

in situ hybridisation
gel electrophoresis
blotting

Question 2

what is in situ hybridisation

Answer 2

hybridisation of a DNA or RNA probe to intact tissue
locates its complementary strand by autoradiography
used to detect mRNA or DNA

Question 3

what is a dna or rna probe

Answer 3

stretches of single-stranded DNA
used to detect the presence of complementary nucleic acid sequences

Question 4

what is autoradiography

Answer 4

imaging technique that uses radioactive sources contained within the exposed sample

Question 5

FISH

Answer 5

florescence in situ hybridisation
used to identify specific sequences in whole tissue with mRNA probes
probes cannot be seen so labelling is needed

Question 6

gel electrophoresis

Answer 6

separates dna fragments by size
uses an electric current - causes dna to move to the positive charge

Question 7

how is dna visualised in gel electrophoresis

Answer 7

uses a visible intercalating molecule that binds in between base pairs - gives dna a colour

alternative - radioactive labelling

Question 8

how is the size of a fragment measured in gel electrophorsis

Answer 8

compared with a fragment of known size

Question 9

what is blotting

Answer 9

hybridisation technique that allows detection of mrnas and dnas

Question 10

what is southern blotting

Answer 10

involves transfer of dna from a gel to a membrane
followed by detection of specific sequences by hybridisation
with a labelled probe

Question 11

what are the three types of dna sequencing

Answer 11

sanger sequencing, next gen. sequencing, PCR

Question 12

what is sanger sequencing

Answer 12

cloned dna are rapidly sequenced by dideoxy chain termination
uses dideoxynucleoties triphosphare (ddATP) to block further growth - terminates dna synthesis

eventually there will be a mixture of preamture daughter fragments ending at every ddNTP - N is replaces with each ATGC

Question 13

limitations of sanger

Answer 13

difficult to parallise
cloning is time consuming
large amounts of dna are needed

Question 14

advantages of sanger

Answer 14

can be done in a single test tube
multiple primers can be used to see overlapping parts of the genome
modern - reusable, can be used for multiple samples
-> cheaper

Question 15

what is a primer

Answer 15

a single stranded nucleic acid molecule
with 3’ OH to initiate dna pol replication of a paired template

Question 16

what is integration in next gen sequencing

Answer 16

the efficiency of sequencing pipeline

ssDNA attaches to silicon plate

covered with probes in DNA pol, primer

DNA pol adds one nucleotide at a time as they are all terminating nucleotides

sequence can then be analysed

Question 17

advantages of Next gen sequencing

Answer 17

no dna library needed - in sanger it is one sequence per tube

in next gen - many sequences are done at the same time by finding the first nucleotide for all sequences then the second and so on.

complementary sequences in overlapping regions can be analysed

Question 18

adaptor sequences

Answer 18

fragments are modified
added to dna fragments in next gen sequencing
used to unwind dna and create ssDNA - adaptor put on silicon like a chip -
primer to adaptor is added
dna pol with all terminating nucleotides used

Question 19

what is pcr

Answer 19

allows for amplification of a desired sequence

using primers that anneal to sequence of interest

Question 20

what is rt pcr

Answer 20

reverse transcriptase pcr
uses reverse transcriptase to convert rna to dna

Question 21

what is real time or quantitative pcr

Answer 21

detects the products of pcr amplification during synthesis

+more sensitive

Question 22

what is thermostable dna polymerases

Answer 22

dna pol that can withstand multiple cycles of template denaturation

thermocycler used to amplify dna

dna pol needed to create a copy - two primers on opp sides

Question 23

northern blotting

Answer 23

hybridisation technique
that allows detection of mRNAs

needs transfer of rna frrom a gel to a membraen
followed by detection of specific sequences with a labelled probe

Question 24

what are dna microarrays

Answer 24

used to detect levels of all expressed genes in a sample

dna microarrays have known dna sequences on a small chip

used to determine if a sequence has a mutation like in BRCA

Question 25

what is genome wide transcription analysis

Answer 25

performed using labelled cDNA from experimental samples

hybridised to a microarray containing sequences from all ORFs of the organism

Question 26

competitive hybridisation

Answer 26

red and green cDNAs to the microarray is proportional to the abundance of each mRNA in the two samples

Question 27

what are expression vectors

Answer 27

used to introduce a gene into a target cell

contain promoters and ribosome binding sites
- shine delgano sequence
allows for efficient transcription and translation of nay cloned dna

most vectors use the lac operon promoter

transcription from the expression vector is induced by addition of a lactose analogue

Question 28

what is the shine delgano sequence

Answer 28

enables initiation of protein synthesis by aligning the ribosome with the start codon

Question 29

SDS gel electrophoresis

Answer 29

proteins are separated accroding to size
SDS is a negatively charged detergent - gives all proteins a negative charge
amount of SDS molecules a protein can bind to is proportional to its size
specific proteins can be detected by blotting with antibodies

Question 30

western blot

Answer 30

proteins separated by size on SDS gel
transferred to nitrocellulose membrane
detected using an antibody with is coupeld to a fluorophore

Question 31

nitrocellulose membrane

Answer 31

a matrix
used for high binding affinity to proteins