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Definitions:
Restriction endonuclease: An enzyme which recognises a palindromic sequence of DNA. It cuts DNA leaving sticky-ends or blunt-ends.
Recombinant DNA: DNA from two or more different organisms
Oligonucleotide: Short sequence of nucleotides complementary to a specific sequence of DNA.
Sequencing: Mapping out the entire genome, or the section of a genome of a particular organism. This can be achieved in many different ways such as Sanger sequencing/Illumina sequencing and modern day mini-Ion sequencing.
Binary fission: The way in which bacteria asexually reproduce to make 2 new bacteria.
Sticky end: A region of ssDNA on the end of a region of dsDNA. This can also be viewed as an overhang.
In-vivo gene cloning + recombinant DNA Technology:
Cloning a gene in vivo can be difficult to understand. First grasp that cloning in vitro is PCR (see below). This is using a dividing organism in order to amplify a gene/protein product.
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Plasmids from bacteria are cut using a restriction endonuclease, which cuts the plasmid leaving sticky end overhangs.
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The same RE are used to excise the desired gene. Since the same RE is used, it cuts out the gene with complementary sticky ends to the plasmid.
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The gene is then ligated into the plasmid using DNA ligase, which seals the nucleotides together with phosphodiester bonds.
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Ca2+ shock and electrical shock stimulate the bacteria to take up the plasmid and divide by binary fission, increasing the amount of the gene as well as synthesising the protein product.
Identifying successful candidates:
By inserting the desired gene into a pre-existing gene that the bacteria has for antibiotic resistance, you can see which bacteria have taken up the gene as they will die (if the desired gene inserted into antibiotic resistance gene). Pressure plating can be used to transfer colonies and grow the sample.
PCR:
-DNA is heated to 95 degrees Celsius, double strand melts to form two single strands.
-Mixture cooled to 60 degrees Celsius to allow primers to anneal.
-Mixture heated to 72 degrees Celsius to allow Taq polymerase to synthesise two new strands.
-Cycle repeats
This occurs in a thermocycler- a machine specifically designed to cycle these temperatures. It contains the primer mixture, free floating nucleotides and Taq pol.
RT-PCR:
Reverse Transcriptase Polymerase Chain Reaction allows a complementary strand of DNA to be made from RNA. This single stranded DNA template of the RNA can then be made into a double-helix using DNA polymerase. In this way it allows for the synthesis of working double-stranded genes.
Reverse Transcriptase:
Reverse transcriptase is an enzyme that synthesises a complementary strand of DNA from a single strand of RNA. It is therefore the reverse of transcription because we make DNA from RNA, not RNA from DNA. Viruses have RT so that they can synthesise a DNA strand once they have deposited their RNA genome into the host cell. By making DNA and then integrating it into the host genome using an integrase enzyme, the virus goes undetected and starts to use the host cell as a protein factory.
Gene markers/probes:
These are short oligonucleotide sequences which bind by complementary base pairing to single stranded DNA. Picture a specific known mutated sequence ACCG.
A
C
C
G
T
G
G
C
Probe
Probe:
This single stranded sequence is combined with fluorescent labelling for visualisation of the mutated gene being present.
The probe is made by taking a double stranded DNA sample of the mutated sequence and then melting it (breaking hydrogen bonds between base pairs across double-helix.
Genetic fingerprinting: The non-coding regions (introns) of DNA can often be misunderstood. Whilst on the surface it may appear that non-coding regions of DNA are 'junk', when looked at closely, it has been noted that satellite regions of DNA express VNTRs (variable number tandem repeats), where discrete sequences of DNA are repeated. Since each individual has their own unique VNTR profile, by screening people for these genetic markers of individuality genetic fingerprinting has applications in crime and paternity testing.
Gel Electrophoresis separates DNA fragments based on size using electricity.
First, the sample is digested using many different restriction endonucleases. This leaves DNA in small fragments. When run on a GE plate, each person will have unique bands correlating to their DNA makeup. Small fragments move quicker towards the positive terminal, whilst large fragments migrate slowly.
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