Gene Technologies Flashcards
What is Recombinant DNA
- form of genetic engineering
- involves the transfer of fragments of
DNA from one organism to another via isolating fragments of interest
Why is genetic information transferable between species?
Genetic code is universal therefore the same amino acid code for the same amino acids in all living things
What are the 3 methods to create fragments of DNA?
- Reverse Transcriptase (enzyme)
- Restriction endonucleases (enzyme)
- Gene machine
What is Reverse Transcriptase?
This process uses reverse transcriptase to make DNA copies of mRNA that was transcribed from the gene of interest
What is the process of Reverse Transcriptase?
- A cell that naturally produces protein of interest is selected
- Once mRNA is isolated reverse transcriptase joins DNA nucleotide w/ complementary base pairs to the mRNA sequence
- the mRNA is used as template to make the single stranded complementary DNA
- DNA polymerase is then used to convert the single stand of cDNA into double stranded which contains the desired code for the gene
Advantage of Reverse Transcriptase
- mRNA is present in cell is from transcribed genes → so lots of the mRNA of interest is available to make cDNA
Disadvantage of Reverse Transcriptase
- more steps, so more time consuming & more difficult
What is restriction endonucleases?
- this process uses the enzyme restriction endonucleases to cut up the DNA
- RE naturally occur in bacteria as a defence mechanism (if there is foreign DNA inside a bacteria, the enzyme will cut it up so it doesn’t replicate and cause harm)
What is the process of restriction endonucleases?
- RE will separate the 2 strands of DNA at a specific base sequence by cutting the sugar-phosphate backbone to give sticky ends or **blunt ends **
BLUNT ENDS:
- caused be being cut straight across
- do NOT result in exposed bases
STICKY ENDS
- caused by cutting the DNA staggered
- result in exposed DNA bases
- palindromic (read the same front to back) - At sticky ends, the exposed base pairs can be aligned to exposed bases of the organism you want to insert it in → makes it easier to insert the desired gene into another organisms DNA
can easily form H bonds w/ complementary base sequences on other pieces of DNA that have been cut w/ same restriction enzyme
Advantage of Restriction Endonuclease
- Sticky ends on DNA fragment make it easier to insert in the DNA fragment when creating recombinant DNA
- can select enzymes that’ll cut at palindromic recognition sites to give sticky ends
Disadvantage of Restriction Endonucleases
- Still contains introns as its cut from the original DNA–> an extra step is required to remove introns
What is the gene machine?
using computers to generate the nucleotide sequence to produce the gene
What is the process of the gene machine?
- Scientists examine the protein of interest to identify the amino acid sequence
- The DNA sequence is entered into the computer (this checks that he DNA being created is safe and ethical to produce)
- The computer can create small sections of overlapping single strands of nucleotides that make up the gene (aka oligonucleotides)
- Oligonucleotides can then be joined to create the DNA for the entire gene
- Polymerase Chain Reaction can be used to make lots of copies of the gene and make it double stranded
Advantages of Gene Machine
- very quick, very accurate
- can design exact DNA fragment you want with sticky ends, DNA probes & won’t have introns
Disadvantages of Gene Machine
- need to know the sequence of amino acids or bases
- time consuming
What is a DNA probe?
Short, single stranded prices of DNA with complementary base pairs.
What makes DNA probes identifiable?
- Radioactively labelled probes
-Fluorescently labelled probes
?
1) Design DNA Probe w/ complementary sequence to allele being screened for
2) patients double stranded DNA is treated to separate and become single stranded
3) mis DNA with the probe binds can be identified
4)he ste at which the
use of gene techinolgies
- allows screening for potential genetic disorders
- can identify presence of oncogenes
- can screen for multiple disease simultaneously using an array where multiple DNA probes are present
How to locate alleles of genes?
1) must know DNA base sequence in order to design the probe
2) Could use DHNA sequencing techniques
3) produce DNA fragments 0 enzyme methos or gene machine
4)amplify- in vitro or in vivo cloning
5) add label- radioactive nucleotide or fluorescent label
6) presence of label indicates allele of intertest is present in patient DNA
After hybridisation , DNA is washed so any unbound probes are washed away
why do we use gene technologies?
- drug screening -some drugs are more/ less effective depending on genotype
- cam help determine effective dose
- saves money
- increased effectiveness
- increase safety
-less waste
