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genetics > molecular bio techniques I/II/III > Flashcards

molecular bio techniques I/II/III Flashcards

1
Q

recombinant DNA technology -

A
  1. restriction enzymes cut DNA into pieces (isolate sections of DNA)
  2. make multiple copies of DNA - insert isolated DNA into cloning vectors (plasmids) so they can be replicated and create a recombinant DNA molecule
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2
Q

diff restriction enzymes recognise diff ….. sequences of DNA and cuts both strands of sugar-phosphate backbone

A

nucleotide

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3
Q

how do restriction enzymes protect bacteria

A

protect bacteria against viruses by cutting viral DNA

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4
Q

restriction enzymes sequence forms a palindrome -

A

sequence reads the same on both strands in 5’ to 3’ direction

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5
Q

restriction enzymes leaves cohesive/blunt ends that are …

A

complementary, less useful in cloning

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6
Q

how do bacteria prevent restriction enzymes cutting their own DNA

A

making a methylase enzyme that is sequence specific
-adds methyl group at the restriction site to stop it from being cut

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7
Q

if genome has many … restriction enzyme cuts less frequently in that genome

A

GC

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8
Q

large size fragments - restriction sites close or far away from each other

A

far away from each other

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9
Q

to find out where restriction enzymes cut in DNA

A

restriction enzyme maps made by cutting DNA with individual restriction enzymes
-fragments separated using electrophoresis - separate according to size
-can identify size of fragments by comparing how far they move in gel compared to fragments of known sizes

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10
Q

to tell us position of restriction enzymes in each fragment

A

single restriction fragment isolated and cut with 5 different restriction enzymes to see if these enzymes cut within DNA region

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11
Q

restriction maps used in - (2)

A

forensics or genetic testing
-detect differences between individuals or sites of mutations
-if DNA sequence changed at a restriction site (mutation) - restriction map changes so size of restriction fragments changes

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12
Q

why do plasmids have an origin of replication

A

so replication happens independently of bacteria

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13
Q

why do plasmids have antibiotic resistance genes

A

allows selective growth of bacteria that contain plasmids

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14
Q

plasmids used for DNA cloning have MCS

A

multiple cloning sequence - individual restriction sites clustered together
WHERE DNA INSERTS CAN BE CLONED

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15
Q

2 types of plasmid - cloning plasmid -

A

cloning genes (storing DNA of plasmid)

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16
Q

2 types of plasmid - expression plasmid -

A

allow gene expression of cloned genes to produce large amounts of encoded protein eg. insulin

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17
Q

creating recombinant DNA molecule

A

restriction fragment and plasmid cut with same restriction enzyme
-same cohesive ends so complementary base pairing forms H bonds
-ligase enzyme forms phosphodiester backbone to form recombinant bacteria
-free 3’OH and phosphate covalently joined by DNA ligase

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18
Q

uses of cloned DNA:
1.creating genomic library -

A

clone and piece together genomic DNA to allow mapping and sequencing of genes in the genome

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19
Q

uses of cloned DNA:
2.

A

identify changes in genome associated with particular phenotypes/diseases
-characterise how genome is organised

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20
Q

plasmids allow cloning of small/large fragments

A

small up to 20kb

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21
Q

cloning vectors used to clone larger fragments

A

bacteriophage vectors
cosmids
artifical chromosomes

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22
Q

to create CDNA (complementary DNA)
CDNA library -

A

isolate mRNA where introns are removed and convert back to DNA to express eukaryotic proteins
-using enzyme reverse transcriptase
-mRNA digested
-DNA pol creates 2nd strand from single stranded DNA
-DNA ligated into a cloning vector eg. plasmid

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23
Q

CDNA for different tissues contain same/diff genes

A

different genes as different tissues express different genes

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24
Q

what is a CDNA library

A

large collection of plasmids each containing a single CDNA

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25
clones in CDNA library lack (2)
introns regulatory sequences
26
DNA cloning to form recombinant DNA allows production of
therapeutics eg. insulin
27
DNA sequencing is used to
sequence DNA clones -use DNA pol to copy single stranded DNA -dideoxy nucleotides stop DNA polymerase copying DNA -after incorporation of dideoxy nucleotide, DNA pol cannot extend chain of nucleotides
28
what is dideoxynucleotide chain-termination sequencing (sequencing of DNA)
incorporation of a dideoxynucleotide - DNA pol cannot extend chain of nucleotides -rapid sequencing of large amounts of DNA
29
DNA normally replicated using dNTPs (deoxynucleotides)
DNA pol joins nucleotides by forming phosphodiester bond between phosphate on 1 molecule and OH on previous molecule
30
DNA replication - dNTPS DNA sequencing - uses
ddNTPs (dideoxynucleotides) - no OH on 3' carbon - has a H DNA pol can't attach more nucleotides as there's no 3 OH' to form phosphodiester bond SO AS SOON as ddNTP is incorporated DNA pol stops extending chain of nucleotides
31
to sequence a DNA fragment - .. parallel sequencing reactions are done with diff dideoxynucleotides - A,C,G,T
4
32
products from each sequencing reaction are run in separate lanes on a ....
polyacrylamide gel
33
polyacrylamide gel allows
separation of DNA fragments differing in length by a single nucleotide -smaller fragments move faster, larger fragments move slower
34
automated DNA sequencing
instead of using radioactivity to see all fragments, make different dideoxynucleotides diff colours -each ddNTP labelled with a unique flourescent marker -identify which ddNTP was added by identifying fluorescent colour
35
sequencing technologies (3)
-original dideoxy sequencing (chain termination sequencing) is very slow -flurorescent capillary dideoxy sequencing is faster -next generation sequencing - adding nucleotides on microchips - can sequence millions of DNA fragments
36
genome is 99.9% same but can identify ...... differences between individuals
single base pair differences (SNPs)
37
PCR is used to
isolate and amplify specific DNA fragments without needing cloning vectors or restriction enzymes -very sensitive - can amplify DNA fragments from small amounts of DNA
38
PCR steps are repeated continuously using a .... to amplify DNA, no. of copies of fragment is .... in each cycle
thermocycler doubled
39
steps of PCR (3)
1. denaturation - reaction heated to 95 degrees to denature DNA into single strands 2. primer annealing - reaction temp reduced to 45-68 degrees to allow primers (short nucleotide sequences) to bind to complementary sequences - 2 primers - forward and reverse primer 3. primer extension - DNA inbetween primers is amplified. Reaction temp raised to 72 degrees to allow Taq polymerase to synthesise DNA
40
why is taq polymerase used in PCR
doesn't get denatured when heated to 96 degrees
41
PCR product - amplified DNA fragments separated by
gel electrophoresis
42
PCR product size =
amount of DNA between the primers
43
limitations of PCR - can only amplify .... sequences of DNA
short
44
limitations of PCR - info of nucleotide sequence of target DNA must be known to make ....
primers
45
why is PCR being sensitive a limitation
little bit of contamination of sample DNA can cause problems
46
what is RT-PCR
reverse transcription PCR -study gene expression by examining mRNA production by cells/tissues -reverse transcriptase converts mRNA to cDNA, amplify cDNA
47
what is QPCR
quantitative PCR - quantify amplification reactions as they happen in real time to identify amount of DNA in a sample
48
PCR can be used for .... testing
paternity
49
nucleic acid hybridization allows
identification of DNA or RNA that match a specific sequence
50
what conditions needed to separate DNA strands
heating or alkali conditions
51
what conditions needed to bind complementary DNA strands
cooling or neutralisation
52
nucleic acid hybridisation either have: (2)
-CDNA library - has lots of plasmids each inserted with a different CDNA - to find which plasmid has the DNA sequence that matches the specific sequence -DNA fragments to find which DNA fragment has DNA sequence that matches specific sequence use ssDNA as a probe to identify specific DNA fragments or clones that have sequence complementary to probe DNA
53
DNA probe preparation - why is DNA probe labelled and using what
so DNA probe can be detected -using DNA polymerase to incorporate labelled dNTPs -dNTPs are labelled by making them radioactive using 32P or by attaching fluorescent molecule
54
steps of DNA probe preparation
1. template DNA used to make the probe. template DNA is denatured, short random primers annealed to DNA 2. Klenow fragment - DNA pol, makes a copy of template DNA and adds labelled dNTPs. probe DNA - labelled DNA fragments copied from the template DNA 3. labelled DNA is denatured to form single stranded probe DNA
55
using nucleic acid hybridisation to identify clones in a CDNA library containing specific DNA sequences =
1. bacterial colonies with plasmids with individual CDNA clones are grown on an agar plate 2. colonies transferred onto DNA binding membrane 3. bacteria lysed and DNA denatured using alkali 4. membrane placed in a heat-sealed bag with solution containing labelled probe. membrane hybridised with labelled DNA probe. SSDNA from plasmid hybridises with labelled denatured DNA 5. wash membrane to remove excess radioactive probe bound. x ray film placed over, dots show hybridisation of probe to 1 colony from original plate 6. cells picked from the colony that are hybridised to the probe and transferred to medium for growth
56
southern blot method to identify which DNA fragment has a specific gene after genomic DNA is cut with particular restriction enzyme =
1. DNA fragments separated by gel electrophoresis and denatured by soaking gel in alkali so double stranded DNA become single stranded and attach to membrane 2. DNA fragments are attached to membrane 3. Labelled DNA probe is added to membrane with fragments bound 4. DNA probe hybridises to complementary DNA fragment on membrane so position of the complementary DNA fragment can be identified as it is labelled by the probe
57
northern blot hybridisation to identify ... molecules containing specific sequences
RNA -identify if a specific gene is transcribed into mRNA in a particular tissue -identify which tissue expresses gene at certain times
58
steps of northern hybridisation =
1. extract mRNA from tissue and separate by size on agarose gel 2. mRNA is transferred directly to membrane - doesn't have to be denatured as it is single strand 3. labelled DNA probe is hybridised to RNA on membrane 4. single stranded DNA probe hybridises to complementary RNA molecules
59
what is in situ hybridisation
gene is expressed in situ - hybridise probe directly to RNA without blotting -probe hybridised to mRNA transcript in situ - where transcript is made -digoxygenin or fluorescently labelled probes are used
60
what are DNA microarrays
modern devices which use nucleic acid hybridisation to measure genes expressed -can identify all genes expressed in a sample -mRNA converted to cDNA, fluorescently labelled and used as a probe
61
DNA microarray has oligonucleotides - what is this
small sequence
62
DNA microarray method
1. extract mRNA from cells 2. convert mRNA to CDNA and label it 3. hybridise with SS probes and will bind if complementary
63
microarrays use nucleic acid hybridisation to allow study of
gene expression from a genomic perspective
64
what is transcriptome and study of transcriptomes
set of transcripts present in a sample - study of transcriptomes - transcriptomics - allows study of which genes are expressed under any type of conditions
65
what are comparative microarrays
DNA microarrays can be used to compare gene expression in 2 samples -compare gene expression in normal and diseased cells
66
commonly used as a label when making probes for DNA hybridisation (3)
digoxygenin fluorescent tag radioactive label
67
which nucleic acid hybridisation technique requires microscopy
in situ hybridisation
68
which of the restriction enzymes produce blunt ends when they digest DNA
AIuI
69
not commonly found in a cloning vector
promoters, transcription initiation site, translation initiation site
70
cloning vector or expression vector can only be plasmids
expression vector
71
the transcriptome =
total mRNA and all non-coding RNAs in a particular sample

genetics (10 decks)

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