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Human Molecular B > Week 8 -molecular Genetic Techniques > Flashcards

Week 8 -molecular Genetic Techniques Flashcards

1
Q

What sort of techniques are used in DNA cloning?

A

Recombinant DNA technology

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2
Q

What is the purpose of DNA cloning?

A

Allows preparation of large numbers of identical DNA molecules, often genes

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3
Q

What do DNA clones allow for?

A

Allows detailed studies of the structure and function of a gene at molecular level

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4
Q

What is Recombinant DNA ?

A

any DNA molecule composed of sequences derived from different sources

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5
Q

Recombinant DNA technology summary

A

Vector and fragment (e.g. gene) Recombinant DNA Replication of Recombinant DNA within host cells Isolation, sequencing and manipulation of purified DNA fragment

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6
Q

What are nucleases?

A

Hydrolyse an Ester bond within a phosphodiester bond

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7
Q

Endonucleases

A

Nucleases that cleaves phosphodiester binds within a nucleic acid chain It may be specific for RNA or for single-stranded or double-stranded DNA

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8
Q

Exonuclease

A

Nuclease that cleaves phosphodiester bonds one at a time from the end of a polynucleotide chain It may be specific for either 5’ or 3’ end of DNA or RNA

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9
Q

Restriction endonucleases

A

Bacterial enzymes that recognise specific short sequences of DNA (4-8bp) called restriction site and cleaves both DNA strands Usually palindromic sequences Are crucial in facilitating the production of Recombinant DNA molecules

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10
Q

Type II endonucleases

A

Cut DNA at defined positions close to or within their recognition sequences

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11
Q

What are Isoschizomer?

A

Pairs of restriction enzymes specific to the same recognition sequence

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12
Q

For each restriction enzyme, what does bacteria produce which has the same DNA recognition sequence?

A

Modification enzyme

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13
Q

What is modification enzyme usually?

A

Methyltransferase

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14
Q

Can methylated recognition sequence be cleaved by a recognition enzyme ?

A

No

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15
Q

How is a bacterium’s own genome/DNA protected from cleavage?

A

Modifying it by methylation at or near potential cleavage sites

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16
Q

What does cloning a fragment of DNA require?

A

Specifically engineered vector

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17
Q

Cloning vector

A

DNA that can be used to propagate an incorporated DNA sequence in a host cell

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18
Q

What do vectors Contain?

A

Selectable markers and Replication origins to allow identification and maintenance of vector in the host

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19
Q

What is multiple cloning site?

A

Synthetically generates sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating Recombinant molecules

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20
Q

What are examples of Multiple cloning site?

A

SphI, PstI, BamHI

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21
Q

What can Restriction endonucleases be used to do?

A

cleave DNA into defined fragments

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22
Q

Inserting DNA fragments into Vectors

A

DNA fragments with sticky or blunt ends can be inserted into vector DNA with aid of DNA ligand e.g. t4 DNA ligases.
Uses 2 ATP to convert 2AMP + 2PPI

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23
Q

Why is it usual to use 2 different restriction enzymes?

A

Directionality Stop recircularisation is the plasmid

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24
Q

What is the insert to vector molar ratio?

A

5- or 10 to 1

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25
Q

What ends May DNA fragments have?

A

Sticky or blunt ends

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26
Q

What is required to insert DNA fragment into vector DNA ?

A

DNA ligase e.g. t4 DNA ligase Use 2 ATP converting to 2MP + 2PPi

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27
Q

What happens to fragments restricted with same endonuclease?

A

They have complimentary ends covalently joins the ends of restriction fragment and vector DNA

28
Q

What is transformation?

A

The acquisition of new genetic material by incorporation of added exogenous, non-viral DNA (e.g. plasmid)

29
Q

What is needed for transformation in lab?

A

Cacl2 Heat shock (42 degrees)

30
Q

Selection of bacteria with antibiotic resistance

A

Bacteria take up plasmid molecule and acquire antibiotic resistance (from plasmid).
This allows for selection of bacteria that harbour the Recombinant plasmid on an agar plate containing relevant antibiotic

31
Q

Application and plasmid purification

A

Colonies are picked from agar plate and grown in liquid broth (with antibiotic) to produce million of bacteria a harbouring the same plasmid plasmid DNA can then be isolated from the methods such as alkaline-lysis based spin column

32
Q

How can plasmid be isolated?

A

Alkaline-lysis based spin column

33
Q

Can cloning create false positives?

A

Yes

34
Q

How can cloning generate false positives?

A

Some plasmids recircularise without an inserted cloned fragment

35
Q

What does blue white selection allow for?

A

Blue/white selection allows the identification of bacteria that contain the vector plasmid WITH an insert (I.e. positive clone)

36
Q

Results of blue white analysis?

A

The insertion of a DNA fragment interrupts the plasmid lac Z gene which means bacteria growing in presence of X-gal are white and not blue The white colonies are selected and used to prepare more plasmid DNA for further analysis

37
Q

Bacteria grown in the presence of X-gal are what colour?

A

White

38
Q

What are the features of a plasmid isolation of DNA limit?

A

High copy number Physical 10kb

39
Q

Feature of a phage isolation of DNA DNA limit?

A

Infects bacteria Via phage packaging 20kb

40
Q

Features of a cosmos isolation of DNA DNA limit ?

A

High copy number via phage packaging 48kb

41
Q

Features of BAC isolation of DNA DNA limit?

A

Based on F plasmid Physical 300kb

42
Q

Features of YAC isolation of DNA DNA limit?

A

Origin + centromere + telomere >1 Mb

43
Q

What can cloning vectors be?

A

Bacterial plasmid, phases, cosmos’s, BAC or yeast artificial chromosome

44
Q

What are shuttle vectors?

A

Can be propagated in more than one type of host cell

45
Q

Expression vectors

A

Contain promoters that allow transcription of any cloned gene

46
Q

What is a DNA LIBRARY?

A

collection of DNA molecule each cloned into a vector

47
Q

What does the set of clones collectively represent?

A

All the DNA sequences in a genome

48
Q

What is the DNA LIBRARY USUEFUL for?

A

Representing the genomic content of simple organisms

49
Q

CDNA libraries

A

Hybridise mRNA with oligo-dT primer Transcribe RNA into CDNA.
Remove RNA with alkali - add poly (dG) tail Hybridise with olive-dC primer Synthesise complementary strands Protect CDNA by methylation at EcoRI sites Ligase cDNA to restriction side linkers cleave with ECoRI Ligate to plasmid

50
Q

Use of reverse transcriptase

A

Used to synthesise a strand of complimentary DNA to each mRNA molecule

51
Q

What is the RNA strand in the RNA/DNA hybrid duplex degraded by?

A

RNaseH or alkali

52
Q

What carries out DNA polymerase?

A

Second strand synthesis

53
Q

Vector and the collection of cDNA are ligated and transformed into what?

A

E.coli

54
Q

Most genomic or cDNA libraries contain how many individual clones?

A

100,000

55
Q

How can molecules of interest be identified in DNA libraries ?

A

Screening

56
Q

How can genomic libraries be screened?

A

Hybridisation of a labelled oligonucleotide probe to complementary sequences can identify specific nucleic acid

57
Q

What is hybridisation?

A

The ability of complimentary single-stranded DNA or RNA molecules to associate specifically with each other via base pairing

58
Q

What is a probe?

A

A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment

59
Q

What are the methods of nucleic acid detection?

A

In-with hybridisation Fluorescence in situ hybridisation

60
Q

What is in situ hybridisation?

A

Hybridisation of a DNA or RNA probe to intact tissue to locate its complementary strand by autoradiography. Used to detect mRNA or DNA

61
Q

FISH

A

Fluorescence in situ hybridisation

62
Q

What are hybridisation methods used to identify?

A

Specific sequences

63
Q

What does gel electrophoresis separate DNA FRAGMENTS BY?

A

Size (shape/confirmation)

64
Q

What charge does DNA migrate towards in electrophoresis?

A

Positive

65
Q

What sort of techniques permit detection of mRNAs and DNAs?

A

Hybridisation techniques

Human Molecular B (13 decks)

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