3.8.4 Recombinant DNA technology, difference in DNA between individuals of the same species can be exploited for identification and diagnosis of heritable condition, genetic fingerprinting, Flashcards
3 Ways of isolating a gene of interest
Using reverse transcriptase
Using restriction endonucleases
Synthesising a gene directly, based on the amino acid sequence (“gene machine”)
5 steps of Recombinant DNA Technology
Isolation
Insertion
Transformation
Identification
Growth/ cloning
how complementary DNA is made using reverse transcriptase
Select a cell type that usually expresses the gene of interest.
The cell’s mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) can be made using reverse transcriptase.
Single stranded cDNA is isolated after hydrolysing the mRNA with an enzyme.
DNA polymerase is used to make a copy of the cDNA by using the cDNA as a template, resulting in a double stranded DNA molecule containing the gene of interest
Why Using a gene made from cDNA is especially useful when transferring a eukaryotic gene into prokaryotic cell
because the cDNA does not contain any introns and prokaryotic cells do not have the ability to remove the intron sequences.
how restriction endonucleases are used to cut DNA into fragments?
Restriction endonucleases are enzymes that cut double-stranded DNA at specific base sequences called recognition sites (restriction sites).
Restriction endonucleases that produce staggered ‘sticky ends’ are most useful for isolating a gene of interest and inserting it into the DNA of another organism.
Their restriction sites are palindromic.
Synthesising a gene directly, based on the amino acid sequence (“gene machine”)
Oligo nucleotides are made in small sections then hybridised to make the gene.
Artificial ‘intron-free’ eukaryotic genes can also be made using a DNA synthesiser.
Preparing the DNA fragment for insertion
A promoter sequence must be present or added in.
This is where RNA polymerase and transcription factors bind, so that the gene can be expressed.
A terminator sequence must be present or added in.
This is where the RNA polymerase is released from the DNA at the end of the gene.
full insertion proccess
The importance of sticky ends
how a DNA fragment can be inserted into a vector?
use of vector
Sticky ends are useful providing both sources of DNA are cut with the SAME restriction endonuclease enzyme. —-Complementary base pairing
Vectors are used to transfer genes from one organism to another
DNA ligase joins the phosphodiester bonds to covalently join the gene and pasid and create a recombinant plasmid
the role of restricted endonuclease
They are used to cut the donor DNA to remove the gene of interest.
They are used to cut the plasmid vector.
Both the plasmid and donor DNA must be cut with the same restriction endonuclease that cuts are the same base sequence recognition site.
This allows the sticky ends of both the plasmid and gene of interest to complementary base pair.
transformation process and outcome
Possible outcomes when mixing the bacteria with the plasmids (vectors)
99% non-transformed - only some bacterial cells (as few as 1%) will take up the plasmids when mixed together
1% successful transformation but the vector does not contain the gene of interest (non-recombinant DNA)
VERY FEW - Successfully transformed AND transgenic
examples of marker genes:
Antibiotic-resistant marker genes
Fluorescent marker genes
Enzyme marker genes
Growth/cloning of the selected bacteria -
select the identified transgenic bacteria and grow them (allow to reproduce) so they produce lots of the desired gene and protein
explain the technique of gel electrophoresis
Gel electrophoresis is a laboratory technique used to separate DNA (or RNA) fragments based on mass / size.
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel.
Smaller samples are less impeded by the gel matrix and hence will move faster through the gel.
This causes samples of different sizes to separate as they travel at different speeds.
how to determine size of dna fragment in gel electrophoresis
- Separate DNA fragments/ladder of known
sizes/lengths; - Compare position/distance/bands with unknown
fragment(s);
DNA probe definition, example
- It is a short single strand of DNA;
- That has bases complementary with DNA/allele/gene;
A DNA probe is a short, single-stranded length of DNA that has some sort of label attached to it that makes it easily identifiable.
Labels can be radioactive (32P, detected using X ray film) or fluorescent tags.
how DNA hybridisation is used to locate specific alleles of genes?
Double stranded DNA is heated so it can be denatured and separated into single strands.
On cooling, the complementary gene probes anneal & hydrogen bond to complementary sequences in the allele of interest.
If the gene probe is complementary to a specific target sequence in the allele, it will hybridise and fluoresce or give off radioactivity if that sequence is present.
