Diagnostics Lec 1 Flashcards

Question

which fragments move further on agarose gel?

Answer 1

smaller fragments

Answer 2

acrylamide gel

Answer 3

Thin capillary tubing contains the gel substance (liquid polyacrylamide for example). Electrophoresis of each DNA sample occurs through the capillary. The DNA sample fragments are labelled with a fluorescent dye for detection. Detection occurs as the DNA fragment passes a particular point in the capillary tube (the detector).

Answer 4

teh amount of fragment (quantity)

Answer 5

indicates the size of the fragments (base pairs)

Answer 6

The electropherogram traces shows a series of peaks. Each peak corresponds to a fragment of fluorescently labelled DNA that is detected when it passes through the detector. The time taken for the fragment of DNA to reach the detector indicates its size. This will be relative to the time taken for size standards to reach the detector.

Answer 7

to selectively amlify a specific piece of DNA

Answer 8

- Recombinant DNA technology Two major strategies: DNA ligase dependent DNA ligase independent

Answer 9

Polymerase Chain Reaction (PCR)

Answer 10

producing large quantities of such a specific sequence of DNA of interest is to introducing it into host cells where it can replicate itself, or be “cloned”, as the host cells divide. This is routinely done in bacteria but can also be done in yeast where much larger fragments of DNA can be replicated

Answer 11

most DNA fragments of interest from many different species are incapable of replicating in bacteria or yeast cells

Answer 12

link DNA fragments to DNA that can replicate in host cells

Answer 13

Bacteriophage lambda, BACs and YACs

Answer 14

The DNA fragment of interest to be cloned is linked to the vector (plasmid) which contains an origin of replication. The linked circular molecule is called recombinant DNA. Competent bacterial cells are most efficiently transformed by circular DNA so recombinant plasmid DNA is easily introduced into bacteria. The recombinant plasmid DNA will replicate in the the resulting bacterial cell clones. The plasmid vectors used replicate, or amplify, to very high copy numbers in bacterial cells and therefore liquid cultures of these cell clones will yield large quantities of the DNA clones when purified.

Answer 15

1) Origin of replication- autonomous replication and propagation - incompatability, mobilisation 2) Encodes ß-lactamase gene- ampicillin resistance - selectable marker to identify and select for bacteria that have been transformed by this plasmid. In this case the selectable marker is the beta-lactamase gene which encodes resistance to the ampicillin antibiotic. 3) Encodes Eco471R- Assists recognition of recombinants (not present on all vectors) - facilitates the identification of bacterial cells that contain a recombinant version of the plasmid which is in effect a suicide gene. The suicide gene encodes a restriction endonuclease called Eco471R. The gene is intact in a non-recombinant version of this plasmid and therefore encodes the enzyme which will now digest the host cell DNA and hence kill the cell. However, recombinant versions of the plasmid have a DNA fragment inserted within the coding region of the Eco471R gene. This disrupts the coding region of the gene and hence it no longer encodes the restriction endonuclease activity. As a result bacterial cells that host a recombinant version of the plasmid survive and are therefore easily identified as the only colonies which develop. 4) Polylinker- Multiple recognition sites for restriction endonucleases - . The polylinker is a sequence of DNA that surrounds the region where genes are inserted into this plasmid. This gives flexibility to allow this fragment to be removed using a combination of restriction enzymes that enable the gene to be transferred to another vector easily

Answer 16

restrition endonucleases and DNA ligases

Answer 17

enzymes that recognise a specific sequence of double stranded DNA and cleave the DNA at a specific site to create two or more double stranded DNA molecules.

Answer 18

DNA methyltransferases

Answer 19

at the recognition sequence for the restriction endonuclease they possess

Answer 20

the enzyme cannot recognise the same nucleotide sequence and therefore the host cell genome is immune from digestion

Answer 21

all recognise a unique sequence; the sequence is recognised in 5’ to 3’ orientation only; the sequence is often a palindrome; position of cuts either create blunt ends or single stranded DNA overhang (sticky ends); sticky ends can either be 5’ overhangs as in examples shown for EcoRI and HindIII or 3’ overhangs

Answer 22

anneal or stick back together (they are compatible)

Answer 23

join together via sticky ends

Answer 24

by the activity of DNA ligases

Answer 25

form a phosphodiester bond between adjacent nucleotides, covalently linking two molecules together when the ends of DNA come together either by hydrogen bonding (base pairing) or random bumping together in the case of blunt ends.

Answer 26

DNA of interest inserted into a plasmid vector then it will be transformed into bacterial cells for amplification

Answer 27

E. coli K12

Answer 28

They need to be sensitive to antibiotics (so those picking up plasmid can be selected for in presence of antibiotic resistance encoded by plasmid) – only cells with vector containing antibiotic resistance gene will grow. They are recombination defective (recA mutation) so no deletions or rearrangements of repetitive DNA sequence can occur. Ecoli K12 have restriction and modification systems. Restriction systems destroy foreign DNA and modification systems protect host cell DNA. Recombinant DNA technology involves introduction of foreign DNA so restriction systems (hsdR mutation) must be disabled

Answer 29

from the surrounding environment

Answer 30

a cells ability to take up foreign DNA from its environment

Answer 31

the actual process of DNA uptake

Answer 32

researchers must induce competence

Answer 33

temporary pores in a cells membrane in order for DNA to pass through

Answer 34

BACK TO SLIDES

Answer 35

low cost versatile different vector choices directional cloning easily done

Answer 36

possible sequence constraints due to presence and/or translation of restriction site

Answer 37

yes, but ligation independent

Answer 38

topoisomerase I

Answer 39

base pairing of T and A nucleotides (T overhangs are created through using TOPO1 enzyme and A overhangs through the use of Taq polymerase which naturally leaves A overhangs at 3' end of PCR products)

Answer 40

cleaves a single strand of double stranded DNA

Answer 41

The basis of TOPO cloning is that the Vaccinia virus TOPO I enzyme only cleaves a single strand of DNA after the sequence CCCTT as shown on this slide. The Phosphate of the T residue and the hydroxyl group of the adjacent residue is shown as P and O in this example. Cleavage of the phosphodiester bond occurs, covalently linking the phosphate of the DNA with a tyrosine amino acid residue of the TOPO I enzyme. This creates a single stranded nick in the double stranded DNA. This process is reversible, as the hydroxyl group of the adjacent nucleotide reacts with the phosphate, breaking the bond with the enzyme and recreating the phosphodiester bond and a double stranded DNA molecule.

Answer 42

gateway cloning

Answer 43

the recombination system that exists between bacteriophage lambda DNA and E.coli host cellular DNA

Answer 44

specific sequences in lambda DNA called attP and E.coli cellular DNA called attB

Answer 45

insertion of bacteriophage DNA into host DNA, bacteriophage sequences being flanked by two new recombination sequences called attL (for left) and attR (for right).

Answer 46

With this system a DNA fragment or gene of interest (GOI) is initially inserted into the polylinker region of a Gateway entry plasmid vector. The DNA fragment is flanked by attP sites (as shown on the left hand side of this slide). The GOI DNA fragment can now be transferred into any other plasmid DNA vector in the Gateway collection that has two attB sites for recombination.

Answer 47

In the example illustrated a GOI is in an entry plasmid DNA vector that also has a kanamycin resistance (KnR) selectable marker. This DNA is mixed in a tube with a destination vector, containing two AttB sites, that has an ampicillin (ApR) selectable marker. The destination vector also has a promoter which would enable expression of the GOI, something not possible with the entry vector shown. A mixture of enzymes, called clonase, are added and they catalyse homologous recombination to take place between the plasmids, mediated by the two attP and attB sites. Homologous recombination results in the transfer of the GOI from the entry plasmid to the destination plasmid. E.coli are transformed with the mixture of recombined plasmid DNA’s and bacterial colonies containing the GOI in the destination vector selected with ampicillin. Again this method is limited to a collection of commercially available Gateway plasmids. Different plasmids in this collection are designed for different purposes. For example you might want to produce the protein encoded by a GOI in bacteria, or in yeast or express the gene in mammalian cells. Each application requires a different plasmid DNA with gene promoters that function efficiently in bacteria, yeast or mammalian cells. Although the system is limited to the collection of plasmids available, it is a very efficient way of transferring a fragment of DNA from one vector to another within the collection.

Answer 48

to assemble multiple sequences together if they have appropriate overlapping regions

Answer 49

T4 DNA polymerase, it is based on homologous recombination

Answer 50

DNA polymerases from either vaccinia virus ( In fusion) or bacteriophage T4 (radiance)

Answer 51

it is sequence independent. Any sequence can, in principle, be inserted into any sequence DNA therefore this technique doesnt have limitations of any of the other methods

Answer 52

the location of restriction endonuclease recognition sequences

Answer 53

double stranded DNA fragments with complimetary sequences located at their ends

Answer 54

to amplify the product with complementary ends

Answer 55

13-15bp in length

Answer 56

A specific vector is used and prepared so that it is linear with specific complementary ends – could be through REs or PCR (long-range PCR for example). The DNA fragments are mixed in a test tube with the DNA polymerase enzyme preparation. The enzyme 3’-5’ exonuclease activity of T4 DNA polymerase removes nucleotides from one strand of the double stranded DNA ends. This creates single stranded DNA ends, which are complementary for the two fragments to be joined. The two fragments of DNA anneal. In E. coli, a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. The annealed DNA is no longer a good substrate for the exonuclease activity. The reaction continues until all DNA fragments are in the annealed configuration. The annealed DNA’s are not covalently linked at this stage as they contain single strand gaps, as indicated by arrows. These gaps are repaired by DNA ligases when linked DNA’s are introduced into bacterial cells by transformation.

Answer 57

anneal, creating linked DNA fragments that have four DNA gaps, indicated by arrows between the adjacent DNA's

Answer 58

circular DNA and teh gaps are repaired by DNA ligases in bacteria, so the DNA's are now covalently linked

Answer 59

The DNA fragments are mixed in a test tube with the DNA polymerase enzyme preparation. The enzyme 3’-5’ exonuclease activity of T4 DNA polymerase removes nucleotides from one strand of the double stranded DNA ends. This creates single stranded DNA ends, which are complementary for the two fragments to be joined. The two fragments of DNA anneal. In E. coli, a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. The annealed DNA is no longer a good substrate for the exonuclease activity. The reaction continues until all DNA fragments are in the annealed configuration. The annealed DNA’s are not covalently linked at this stage as they contain single strand gaps, as indicated by arrows. These gaps are repaired by DNA ligases when linked DNA’s are introduced into bacterial cells by transformation.

Answer 60

Sequence independent but ligase dependent, 3 heat stable enzymes (exonuclease, polymerase, ligase)

Answer 61

The procedure requires 3 heat stable enzymes (5’3’exonuclease, DNA polymerase and DNA ligase) and is performed at 50°C. DNA fragments are created with overlapping sequences in the location where the join is to be made (shown in red and green in the figure). This can be in any position and can be achieved by PCR and is the same as required for the SLIC procedures described previously. Gibson cloning uses T5 exonuclease, which has 5’ to 3’ exonuclease activity which creates 5’ to 3’ sticky ends (making it different to the 3’-5’ exonuclease activity of T4 DNA polymerase). As the sequence of the ends of the two DNA molecules are identical they can stick together (anneal) by hydrogen bonding between complimentary base pairs. The gap between the hydrogen bonded sticky ends and the adjacent strand of DNA is repaired by DNA synthesis, catalysed by DNA polymerase. Finally the two molecules are covalently linked together by a heat stable DNA ligase (Taq ligase).

Answer 62

Rapid, one step reaction Nicks repaired in vitro Enables creation of complex recombinants very versatile - but more expensive than SLIC (because T4 polymerase is cheaper)

Answer 63

TOPO cloning life technology Gateway cloning technology life technology

Answer 64

In-fusion clontech Radiance novagen Gibson cloning

Diagnostics Lec 1 Flashcards

Molecular Diagnostics (93 cards)