Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

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

Week 8 -molecular Genetic Techniques Flashcards

(65 cards)

Start Studying
1
Q

What sort of techniques are used in DNA cloning?

A

Recombinant DNA technology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is the purpose of DNA cloning?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What do DNA clones allow for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is Recombinant DNA ?

A

any DNA molecule composed of sequences derived from different sources

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are nucleases?

A

Hydrolyse an Ester bond within a phosphodiester bond

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Type II endonucleases

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are Isoschizomer?

A

Pairs of restriction enzymes specific to the same recognition sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

Modification enzyme

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is modification enzyme usually?

A

Methyltransferase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Can methylated recognition sequence be cleaved by a recognition enzyme ?

A

No

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

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

A

Modifying it by methylation at or near potential cleavage sites

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What does cloning a fragment of DNA require?

A

Specifically engineered vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Cloning vector

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What do vectors Contain?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are examples of Multiple cloning site?

A

SphI, PstI, BamHI

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What can Restriction endonucleases be used to do?

A

cleave DNA into defined fragments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Why is it usual to use 2 different restriction enzymes?

A

Directionality Stop recircularisation is the plasmid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is the insert to vector molar ratio?

A

5- or 10 to 1

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
What ends May DNA fragments have?
Sticky or blunt ends
26
What is required to insert DNA fragment into vector DNA ?
DNA ligase e.g. t4 DNA ligase Use 2 ATP converting to 2MP + 2PPi
27
What happens to fragments restricted with same endonuclease?
They have complimentary ends covalently joins the ends of restriction fragment and vector DNA
28
What is transformation?
The acquisition of new genetic material by incorporation of added exogenous, non-viral DNA (e.g. plasmid)
29
What is needed for transformation in lab?
Cacl2 Heat shock (42 degrees)
30
Selection of bacteria with antibiotic resistance
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
Application and plasmid purification
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
How can plasmid be isolated?
Alkaline-lysis based spin column
33
Can cloning create false positives?
Yes
34
How can cloning generate false positives?
Some plasmids recircularise without an inserted cloned fragment
35
What does blue white selection allow for?
Blue/white selection allows the identification of bacteria that contain the vector plasmid WITH an insert (I.e. positive clone)
36
Results of blue white analysis?
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
Bacteria grown in the presence of X-gal are what colour?
White
38
What are the features of a plasmid isolation of DNA limit?
High copy number Physical 10kb
39
Feature of a phage isolation of DNA DNA limit?
Infects bacteria Via phage packaging 20kb
40
Features of a cosmos isolation of DNA DNA limit ?
High copy number via phage packaging 48kb
41
Features of BAC isolation of DNA DNA limit?
Based on F plasmid Physical 300kb
42
Features of YAC isolation of DNA DNA limit?
Origin + centromere + telomere >1 Mb
43
What can cloning vectors be?
Bacterial plasmid, phases, cosmos’s, BAC or yeast artificial chromosome
44
What are shuttle vectors?
Can be propagated in more than one type of host cell
45
Expression vectors
Contain promoters that allow transcription of any cloned gene
46
What is a DNA LIBRARY?
collection of DNA molecule each cloned into a vector
47
What does the set of clones collectively represent?
All the DNA sequences in a genome
48
What is the DNA LIBRARY USUEFUL for?
Representing the genomic content of simple organisms
49
CDNA libraries
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
Use of reverse transcriptase
Used to synthesise a strand of complimentary DNA to each mRNA molecule
51
What is the RNA strand in the RNA/DNA hybrid duplex degraded by?
RNaseH or alkali
52
What carries out DNA polymerase?
Second strand synthesis
53
Vector and the collection of cDNA are ligated and transformed into what?
E.coli
54
Most genomic or cDNA libraries contain how many individual clones?
100,000
55
How can molecules of interest be identified in DNA libraries ?
Screening
56
How can genomic libraries be screened?
Hybridisation of a labelled oligonucleotide probe to complementary sequences can identify specific nucleic acid
57
What is hybridisation?
The ability of complimentary single-stranded DNA or RNA molecules to associate specifically with each other via base pairing
58
What is a probe?
A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment
59
What are the methods of nucleic acid detection?
In-with hybridisation Fluorescence in situ hybridisation
60
What is in situ hybridisation?
Hybridisation of a DNA or RNA probe to intact tissue to locate its complementary strand by autoradiography. Used to detect mRNA or DNA
61
FISH
Fluorescence in situ hybridisation
62
What are hybridisation methods used to identify?
Specific sequences
63
What does gel electrophoresis separate DNA FRAGMENTS BY?
Size (shape/confirmation)
64
What charge does DNA migrate towards in electrophoresis?
Positive
65
What sort of techniques permit detection of mRNAs and DNAs?
Hybridisation techniques

Human Molecular B (13 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions