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

molecular bio techniques I/II/III Flashcards

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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
Q

clones in CDNA library lack (2)

A

introns
regulatory sequences

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26
Q

DNA cloning to form recombinant DNA allows production of

A

therapeutics eg. insulin

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27
Q

DNA sequencing is used to

A

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

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28
Q

what is dideoxynucleotide chain-termination sequencing (sequencing of DNA)

A

incorporation of a dideoxynucleotide - DNA pol cannot extend chain of nucleotides
-rapid sequencing of large amounts of DNA

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29
Q

DNA normally replicated using dNTPs (deoxynucleotides)

A

DNA pol joins nucleotides by forming phosphodiester bond between phosphate on 1 molecule and OH on previous molecule

30
Q

DNA replication - dNTPS
DNA sequencing - uses

A

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
Q

to sequence a DNA fragment - .. parallel sequencing reactions are done with diff dideoxynucleotides - A,C,G,T

A

4

32
Q

products from each sequencing reaction are run in separate lanes on a ….

A

polyacrylamide gel

33
Q

polyacrylamide gel allows

A

separation of DNA fragments differing in length by a single nucleotide
-smaller fragments move faster, larger fragments move slower

34
Q

automated DNA sequencing

A

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
Q

sequencing technologies (3)

A

-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
Q

genome is 99.9% same but can identify …… differences between individuals

A

single base pair differences (SNPs)

37
Q

PCR is used to

A

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
Q

PCR steps are repeated continuously using a …. to amplify DNA, no. of copies of fragment is …. in each cycle

A

thermocycler
doubled

39
Q

steps of PCR (3)

A
  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
Q

why is taq polymerase used in PCR

A

doesn’t get denatured when heated to 96 degrees

41
Q

PCR product - amplified DNA fragments separated by

A

gel electrophoresis

42
Q

PCR product size =

A

amount of DNA between the primers

43
Q

limitations of PCR - can only amplify …. sequences of DNA

A

short

44
Q

limitations of PCR - info of nucleotide sequence of target DNA must be known to make ….

A

primers

45
Q

why is PCR being sensitive a limitation

A

little bit of contamination of sample DNA can cause problems

46
Q

what is RT-PCR

A

reverse transcription PCR
-study gene expression by examining mRNA production by cells/tissues
-reverse transcriptase converts mRNA to cDNA, amplify cDNA

47
Q

what is QPCR

A

quantitative PCR - quantify amplification reactions as they happen in real time to identify amount of DNA in a sample

48
Q

PCR can be used for …. testing

A

paternity

49
Q

nucleic acid hybridization allows

A

identification of DNA or RNA that match a specific sequence

50
Q

what conditions needed to separate DNA strands

A

heating or alkali conditions

51
Q

what conditions needed to bind complementary DNA strands

A

cooling or neutralisation

52
Q

nucleic acid hybridisation either have:
(2)

A

-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
Q

DNA probe preparation - why is DNA probe labelled and using what

A

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
Q

steps of DNA probe preparation

A
  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
Q

using nucleic acid hybridisation to identify clones in a CDNA library containing specific DNA sequences =

A
  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
Q

southern blot method to identify which DNA fragment has a specific gene after genomic DNA is cut with particular restriction enzyme =

A
  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
Q

northern blot hybridisation to identify … molecules containing specific sequences

A

RNA
-identify if a specific gene is transcribed into mRNA in a particular tissue
-identify which tissue expresses gene at certain times

58
Q

steps of northern hybridisation =

A
  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
Q

what is in situ hybridisation

A

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
Q

what are DNA microarrays

A

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
Q

DNA microarray has oligonucleotides - what is this

A

small sequence

62
Q

DNA microarray method

A
  1. extract mRNA from cells
  2. convert mRNA to CDNA and label it
  3. hybridise with SS probes and will bind if complementary
63
Q

microarrays use nucleic acid hybridisation to allow study of

A

gene expression from a genomic perspective

64
Q

what is transcriptome and study of transcriptomes

A

set of transcripts present in a sample
- study of transcriptomes - transcriptomics - allows study of which genes are expressed under any type of conditions

65
Q

what are comparative microarrays

A

DNA microarrays can be used to compare gene expression in 2 samples
-compare gene expression in normal and diseased cells

66
Q

commonly used as a label when making probes for DNA hybridisation (3)

A

digoxygenin
fluorescent tag
radioactive label

67
Q

which nucleic acid hybridisation technique requires microscopy

A

in situ hybridisation

68
Q

which of the restriction enzymes produce blunt ends when they digest DNA

A

AIuI

69
Q

not commonly found in a cloning vector

A

promoters, transcription initiation site, translation initiation site

70
Q

cloning vector or expression vector can only be plasmids

A

expression vector

71
Q

the transcriptome =

A

total mRNA and all non-coding RNAs in a particular sample

genetics (10 decks)

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