4.2 Flashcards
Genetic engineering is a
collection of techniques by which genes are altered or transferred from one organism to another
The modified DNA is called:
recombinant DNA
GE involves 3 things:
o Obtaining or isolating the gene to be transferred
o Transferring the gene into the new cells
o Identifying the modified cells
A useful application of GE is the production of human insulin to treat
Type 1 diabetes
Before GE, insulin from pigs was used. This differs from human insulin by only 1 amino acid - sometimes causes an
immune response in diabetics
the gene for human insulin can be inserted into bacterial cells and the bacteria are
grown in large numbers and the insulin is extracted
As human insulin is now used, which is identical in every person, diabetics are unlikely to show an
immune response
Gene for human insulin needs to be obtained - 3 ways to do this:
1-Gene may be CUT OUT of human DNA
2-Gene may be made from mRNA
3- Using DNA bases sequence that codes for human insulin and then making multiple copies (PCR)
1- Cutting out of DNA, 2 stages:
1-Human insulin gene located using a gene probe
2-DNA is cut out using a restriction enzyme
Gene probes are:
specific segment of single-strand DNA that is complementary to a desired gene
Restriction enzymes are:
an enzyme that cuts DNA molecules at a specific sequence of bases called a recognition sequence
-leaving sticky ends
2-Gene may be made from mRNA, e.g. the cells in the pancreas that produce insulin have a large amount of mRNA coding for insulin - this mRNA can be isolated
, 4 steps:
1-Reverse transcriptase is used to make a DNA copy from its RNA
2-Genetic engineers use reverse transcriptase to make a single-stranded DNA copy of the mRNA from the pancreas cells
3-This single-stranded DNA is called cDNA (‘copy DNA’)
4-The cDNA is then made into double-stranded DNA
3-Using DNA bases sequence that codes for human insulin and then making multiple copies (PCR)
- 3 bases code for 1 amino acid
- work out DNA base sequence that codes for human insulin
- use DNA base sequence to synthesise more copies
What is used to transfer the isolated gene into a new host cell?
vector
Most common type of vector =
plasmid
(INSERTING THE DNA INTO NEW CELLS)
Step 1: DNA in the plasmid is cut open using a
restriction enzyme
the restriction enzyme used in step 1 is often the same one used to isolate the new gene, if not, nucleotides are added to the cut plasmid and to the isolated gene so that they have
matching sticky ends
(INSERTING THE DNA INTO NEW CELLS)
Step 2: the gene is then inserted into the cut plasmid using the enzyme
DNA ligase
(INSERTING THE DNA INTO NEW CELLS)
Step 3: the bacterial cells and plasmids are
mixed together
(INSERTING THE DNA INTO NEW CELLS)
Step 4: the bacteria are treated so that many of them take up a
modified plasmid or recombinant plasmid
(USING GENETIC MARKERS)
Some of the bacterial cells do not take up a plasmid, or take up a
non-recombinant plasmid
(USING GENETIC MARKERS)
It is not possible to tell by looking at the cells which one contain the insulin gene, however, the plasmid used contain an
antibiotic resistance gene
(USING GENETIC MARKERS)
The antibiotic resistance gene gives resistance to a specific antibiotic, such as tetracycline, and is called a
genetic marker
(USING GENETIC MARKERS)
The scientists can mix the bacteria with the antibiotic that the marker gene gives resistance to, this will kill all the bacteria that do not contain a
plasmid
(USING GENETIC MARKERS)
Marker genes may also code for
other characteristics that are easy to identify, such as production of a fluorescent protein or a specific enzyme
What enzyme makes a single-stranded piece of DNA using an RNA template?
Reverse transcriptase
What enzyme joins 2 pieces of DNA together?
DNA ligase
What enzyme cuts DNA whenever it recognises a specific base sequence?
Restriction enzyme
