Session 1: Gene Technology Flashcards
What is DNA amplification and how does it occur?
DNA amplification is the process of producing vast quantities of identical DNA from very small DNA samples. This done using a technique called the polymerase chain reaction (PCR).
Describe polymerase chain reaction (PCR).
Amplification of DNA can be carried out using PCR machines called thermal cyclers (shown below). PCR cycles replicate DNA at an exponential rate and can make millions of copies in only a few hours. Step 1: Separate target DNA strands by heating 98°C for 5 minutes. Step 2: Add reaction mix - add primers (short nucleotide strands that provide a starting sequence for DNA replication), nucleotides (A, T, G, and C), and DNA polymerase enzyme. Step 3: Incubate - cool to 60°C and incubate for a few minutes. During this time, primers attach to single-stranded DNA. DNA polymerase synthesises complementary strands. Then, repeat these three steps 25 times (25 cycles) or until enough copies of the target DNA have been produced.
Describe taq polymerase
Taq polymerase is an enzyme isolated from the thermophilic
bacterium Thermus aquaticus. As this enzyme’s optimal temperature is ~75oC, it is able to
function at the high temperatures used in PCR without denaturing (unlike most DNA replication enzymes). Taq polymerase extends the nucleotide chain from the primers –
therefore primers are used to select the sequence to be copied.
Describe Gel Electrophoresis.
Gel electrophoresis separates proteins or fragments of DNA according to their charge and size. Fragments are placed in a gel that is subjected to an electric charge (fragments move to the positive
terminus) DNA fragments are placed in an agarose gel. Proteins are placed in a polyacrylamide gel. Large fragments move slower (impeded by gel matrix). DNA sample fragment size is calculated by comparing against known industry standards. Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography (Southern blotting).
Describe DNA Sequencing.
Sequencing involves the use of chain-terminating dideoxynucleotides (ddNTPs) which lack the 3’-hydroxyl group needed to form a phosphodiester bond. Incorporation of a ddNTP functions to effectively terminate DNA replication. The length of a sequence will reflect the position of ddNTP incorporation. A separate Sequencing reaction is carried out for each of the 4 bases (T, C, G and A). A modified 1% of each base is added to cause termination at random sites. E.g. for a thymine reaction 1% of modified T is added, and for cytosine 1% of C is added. The fragments from the four reactions are separated by electrophoresis and analyzed to determine the DNA sequence. Fluorescent dyes and computer software are now more commonly used for automated sequencing
Describe restriction enzymes.
Restriction enzymes (or endonucleases) are purified forms of naturally occurring bacterial enzymes that are used as “molecular scalpels”. Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites. Some restriction enzymes cut the fragment and leave an overhang as the recognition sites are not at the same location on both strands of DNA. These overhangs are called “sticky ends”. Some restriction enzymes
cut leaving no overhang as the recognitions sites are at the same location on both strands of DNA. These are called “blunt
ends”.
What is DNA profiling?
DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles. Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements called short tandem repeats (STRs). As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA profiles. Only one in a billion people are likely to have an identical DNA profile to someone else, making DNA profiling a useful tool for forensic investigations and paternity analysis.
Describe the procedure for DNA profiling for forensics, etc.
A DNA sample is collected (e.g. from blood, semen, saliva,
etc.) and then amplified using PCR. Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments. Fragment length will differ between individuals due to the variable length of their short tandem repeats. The fragments are separated using gel electrophoresis and the resulting profiles are compared.
Describe Genetically Modified Organisms.
A genetically modified organism (GMO) is a plant, animal or micro- organism whose genetic code has been altered to give it
characteristics that do not naturally occur. This is possible because the genetic code is universal - all living things have the same DNA structure and function. Genetically modified organisms (GMOs) may be created by modifying their DNA in one of three ways: Adding a foreign gene (gene transfer), Altering an existing gene, Delete or ‘Turn Off’ a Gene
What is gene transfer and what are the main steps?
Gene transfer is the process by which genes from one organism are inserted into another organism (horizontal gene transfer). Gene transfer involves four key steps: DNA extraction – Gene of interest and vector (virus or plasmid) are isolated, Digestion – Gene and vector are cut with restriction endonucleases, Ligation – Gene of interest is placed into the vector, Transformation – Recombinant vector inserted into host cells
What is a vector and what are two common examples?
A vector is a molecular vehicle used to artificially carry genetic material into a cell. Two common examples are plasmids and viruses. Plasmids are circular DNA molecules capable of autonomous gene expression. Viruses can inject their genetic material directly into host cells.
Describe ligation.
DNA fragments produced using restriction enzymes may be reassembled (joined) to a vector by a process called ligation using the enzyme DNA ligase. This produces a molecules of recombinant DNA. Steps: Two pieces of DNA are cut using the same restriction enzyme which produces sticky ends. The two different DNA fragments are attracted to each other by weak hydrogen bonds. One end of the foreign DNA is attracted to the remaining sticky end of the plasmid.
