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Bio 114 Lecture 1
Principles of Recombinant DNA Technology
36
Biology
Undergraduate 1
02/20/2013

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Term
DNA from two sources is isolated and .................. with the same .................. endonuclease
Definition

cleaved

restriction

Term
the two types of DNA pair at their .............. ends when mixed together. The enzyme DNA ............ joins them
Definition

sticky

ligase

Term
the plasmid is inserted into the bacterial cell by ................
Definition
transformation
Term
Clones are screened for........ of interest
Definition
gene
Term
which DNA is used for genome mapping and sequencing?
Definition
genomic DNA
Term
which type of DNA contains introns?
Definition
Eukaryotic DNA
Term
why is genomic DNA no use for expressing protein in bacteria?
Definition
because of its introns and very long fragments (>20kb fragments)
Term
what category of DNA is chromosomal DNA?
Definition
Genomic DNA
Term
what is cDNA synthesised from? Which enzyme is used in this process?
Definition
synthesised from mRNA using reverse transcriptase
Term
apart from reverse transcriptase what 2 other things does cDNA need?
Definition

primer

nucleotides:-

dATP dGTP gCTP gTTP

this produces ss cDNA

Term
the second strand of cDNA also requires primer, nucleotides and which enzyme?
Definition
DNA Polymerase
Term
what is the difference between DNA and cDNA?
Definition
cDNA is intron-less. it is an artificial gene
Term
which enzyme degrades RNA when making cDNA?
Definition
RNAse H enzyme
Term
which enzyme adds nucleotides on the 3" end of ssDNA?
Definition
terminal transferase enzyme
Term
small pieces of DNA that can be used as carriers are also known as?
Definition
vectors
Term
which extrachromosomal bacterial DNA often carries antibiotic resistance?
Definition
Plasmids -these are often used as vectors
Term
in relation to symmetry, what do all restriction enzymes have?
Definition
a 2-fold rotational symmetry
Term
restriction enzymes recognize sequences which maybe how long?
Definition
4, 6 or 8 nucleotides long
Term
restriction enzymes are isolated from bacteria. what are their roles in protecting the bacteria from invasion?
Definition
they cut up invading DNA protecting the bacteria from infection
Term
what is the correct name for restriction enzymes?
Definition
restriction endonuclease
Term
what type of DNA will restriction endonucleases cut at defined sites?
Definition

Double Stranded DNA

 

Restriction Endonucleases will not cut ss DNA

Term
if there is a 4 base recognition sequence what is the probability that a restriction endonuclease will cut a piece of DNA?
Definition

1/4 x 1/4 x 1/4 x 1/4 = 1/256

 

on average every 256bp

Term
where on a DNA strand would a restriction enzyme cut?
Definition
the sugar-phosphate backbone
Term
which enzyme joins the sticky ends together to make recombinant DNA?
Definition

DNA ligase

 

DNA ligase joins 5’ phosphate to 3’ hydroxyl in double- stranded DNA 

Term

In a cloning experiment, DNA fragments are joined to a ................ 

Definition

vector

these are 

Relatively small DNA molecules

a single site is usually for chosen restriction enzyme

capable of replicating in host organism

Contain selectable gene (eg antibiotic resistanc 

Term
name 3 hosts that can be used to culture recombinant dna?
Definition

bacteria eg. ecoli but not 0157 strain

Yeast

Animals cells in culture

Term

true or false?

over 3/4 of the host cells take up a plasmid during transformation?

Definition

false

only a fraction of the host cells take up a plasmid during transformation

Term
how can you minimise religation of plasmid vector? (prevent the cleaved sections from rejoining)
Definition

treating linearised vector with phosphotase.

 

this removes phosphate groups and minimises the plasmid from rejoining

Term
name a method used to identify antibiotic resistant cells that also contain recombinant DNA?
Definition
blue- white screening
Term
in blue-white screening, the vector contains a gene that codes for the enzyme B-galactosidase. what is this gene called?
Definition
lacZ
Term
what is X-gal and what does B-galactosidase do to X-gal?
Definition
x-gal is a derivative of lactose and this enzyme converts x-gal into a blue colour
Term
in blue-white screening which colour colonies are the recombinant clones?
Definition

the white ones - this is because

 

no enzyme will be produced as the gene is disrupted by insertion of a DNA frgament

no blue product

white colony identified as recombinant clone

Term
what is a library of clones?
Definition

where a large number of different genomic fragments or cDNAs are inserted into a vector

you get a mixture of recombinant DNAs each with a different insert. each colony derived from a single recombinant DNA molecule = library of clones

Term
how is the DNA of a bacterial cell protected from its own restriction endonucleases?
Definition
by the addition of methyl groups to adenines or cytosines to the sequences recognised by the enzyme
Term
why is genomic library also known as the shot gun approach?
Definition
no specific gene is targeted for cloning and you will have a mixture of recombinant DNA
Term
what is colony hybridisation?
Definition
where a nucleic acid probe (either DNA or RNA) is synthesised complimentary to the gene of interest. this probe is labelled with an isotope or fluorescent tag so we can track it
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