Term
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Definition
| DNA from two different sources combined in vitro |
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Term
| 3 reasons for creating r DNA |
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Definition
create protein product
create multiple copies of gene
insert foreign gens into other organisms to give those organisms a new trait |
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Term
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Definition
DNA from two or more sources incorporated
treat DNA with same restriction endonuclease
cutting DNA by restriction enzymes
The ends of the cut have an overhanging piece of single-stranded DNA.
these stick ends are put together by DNA ligase with links the two molecules into recombinant DNA. |
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Term
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Definition
Used for defense against viruses
recognize and make double stranded cuts in the sugar phosphate back bone of DNA.
called restriction endonucleases.
cuts in between certain sequence of nucleotides- restriction site with is 4-13 nucleotides long
E-coli produces an enzyme named EcoRI that cuts between G and A |
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Term
| 3 Types of restriction enzymes |
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Definition
Type 1: recognize specific sequences in the DNA but cut the Dna at random sites some distance from recognition sequence
Type 2: recognize sequence and cut the DNA within sequence
Type 3: cut DNA at nearby sites, usually 25bp away. |
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Term
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Definition
joins the sugar-phospate backbone bases together
in effect ligase reverses the action of restriction enzymes
ligase is more efficent at closing sticky ends then blunt ends. |
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Term
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Definition
| amplyfies DNA, specific, automated and capable of amplyfying minute amounts of sample. |
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Term
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Definition
initalization
DNA and primers denatured
DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single stands. |
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Term
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Definition
Annealing
Reaction temperature is lowered so that the primers can anneal to the single-stranded DNA templete
polymerase attaches and being DNA synthesis. |
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Term
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Definition
extension/elongation
DNA polymerase synthesizes new DNA strand complementary to the DNA template
95C to get single
55 deg to bind ends of DNA
temp is raised to 72 deg and the DNA polymerase causes the synthesis of new complementary strands
The DNA is doubled at each cycle at the end of the 32 cycles it's been amplified 1 billion times. |
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Term
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Definition
process of moving a gene from one molecule to another
moved into a vector for introduction in to a cell
most common vector is plasmid
an efficient cloning vector has three important characteristics: an origin of replication: replication of vector within the cell
selective markers
one or more unique res sites. |
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Term
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Definition
circular double stranded DNA molecule
often associated with conjugation, a mechanism of horizontal gene transfer
contains orgins of replication- independent replication inside the bacteria
introduced by transformation. |
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Term
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Definition
pBR322,
replication in E.coli
treatment with restriction enzyme lineraizes the vector
contains gene conferring ampicillin and tetracycline
can easily be inserted by transformation. |
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Term
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Definition
| if th foreign gene is inserted in to the gene coding for antibiotic resistance- no resistance to antibiotic. |
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Term
| Uses and limits of plasmid |
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Definition
uses for gene coloning and gene expression
if the sizes of the insert increases, transformation efficiency decreases
plasmids with large inserts are unstable. |
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Term
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Definition
phages contain nucleic acids wrapped in protein coats
after insertion of foreign DNA genome is packaged in to viral capsids and can be used to infect E.coli cells.
1000 times more efficient then the plasmid vector
can not be used for gene expression
cosmids: eukaryotic vectors can be used. |
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Term
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Definition
extreme ends of the lambda page DNA
combo of plasmid vector and COS site which allows target DNA to insert
High transformation
gene expression is possible |
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Term
| selection of host is important as selecting vector |
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Definition
| Host- lack RE and Rec enzymes- better |
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Term
| Methods of inserting genes |
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Definition
Eletroporation
Micro injection
gene gun |
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Term
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Definition
cell mixed with rDNA and exposed to brief pulse of high voltage electricity
plasma membrane-temporarily permable
DNA taken up cells grown in media. |
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