What is a DNA primer? What are they marked with?
- short DNA sequences complementary to the DNA adjacent to the STR
- primers are marked with fluorescent tags
What is a nucleotide?
- a nucleotide is an organic molecule with a basic composition of a nitrogenous base, pentose sugar and phosphate
- it is the basic building block of nucleic acids (RNA and DNA)
What is DNA polymerase?
- DNA polymerase is an enzyme that synthesises DNA by adding nucleotides one by one to the growing DNA chain
What does the process annealing refer to?
- annealing is the process of joining of single-stranded DNA or RNA by hydrogen bonds to form a double-stranded polynucleotide
- when two complementary strands of DNA or RNA have paired with each other or a synthetic primer
What does PCR stand for? What is used to make?
- PCR stands for polymerase chain reaction
- polymerase chain reaction is used to make (numerous) new copies of all the fragments of DNA present (‘amplifying’ the DNA)
- often only small amounts of DNA are available for forensic analysis so the DNA fragments loci are copied to get enough DNA to make a profile
What does STR stand for? What are STR’s? How many base pairs does it contain and how many times can it be repeated?
- STR stands for short tandem repeats or satellites
- STR’s are sections of repetitive DNA where a short sequence is repeated multiple times (STR’s are sequences or repeated bases)
- An STR can contain from 2 to 50 base pairs and can be repeated from five to several hundred times
What are introns? Where are they found?
- introns are the non-coding regions of the gene that do not contain codons needed to make the final protein (intragenic regions)
- introns are found between exons
What are exons?
- exons are the coding regions of the gene (expressed regions)
- they contain the codons that are later read to make protons
What is southern blotting used to do? What is it named after?
- southern blotting (named after Ed Southern who developed the technique) is used to transfer the fragments to a more resilient nylon or nitrocellulose membrane
What happens to the fragments during southern blotting?
- during southern blotting process the fragments maintain their positions relative to each other and are denatured into single strands exposing base sequences
- DNA denatures in alkali solution
What is a DNA probe? What can the probes be labelled with and why?
- a DNA probe is a short, single-stranded section of DNA that is complementary to the target DNA sequence
- the DNA probe can be labelled with a radioactive marker or a fluorescent marker to help see the location of the DNA fragments
What is a DNA ladder/ marker added to?
- a DNA ladder or marker is sometimes added to the gel (in gel electrophoresis)
What does gel electrophoresis separate? What does this depend on?
- DNA fragments produced by restriction enzymes or PCR can be separated by gel electrophoresis depending on their size
What are three ways in which a body can be identified?
a body can be identified by personal identification, fingerprinting or dental records
What are three ways in which a DNA sample can be obtained?
- cells obtained in a cheek swab
- white blood cells in a blood smear
- bone marrow in a skeleton
- sperm left after a sexual assault
How is DNA extracted (think back to kiwi)?
- tissue sample is physically broken down in a buffer solution including salt and a detergent to disrupt the cell membranes
- small suspended particles, including DNA, are separated from the rest of the cell debris by filtering or centrifuging
- protease enzymes are incubated with the suspension to remove proteins
- cold ethanol is added to precipitate out the DNA
- several stages of then washing the DNA in a buffer solution then follow
How is a DNA profile made?
- obtaining the DNA
- Cut the DNA into fragments using restriction enzymes
- Amplifying DNA present in the sample (polymerase chain reaction)
- separate out the fragments into a characteristic pattern (DNA fingerprint) using gel electrophoresis
- southern blotting to transfer DNA onto membrane
- disclosure - show the presence of the DNA using a DNA probe or staining. Loading dye can be used to show progress of DNA through gel
What are restriction enzymes? What are they used for in labs?
- restriction enzymes (aka restriction endonucleases) are bacteria produced enzymes that are used to cut DNA into smaller fragments only where their specific restriction sequences occurs
- they are used as targeted ‘scissors’ in the lab
How is a DNA profile made from a small sample of DNA? (in own words)
- multiple copies of DNA made using polymerase chain reaction (PCR)
- step 1: 90 degrees to 95 degrees
step 2: 50 degrees to 65 degrees
step 3 70 degrees to 80 degrees - restriction enzymes used to cut the DNA into smaller fragments
- gel electrophoresis used to separate the fragments
- DNA is placed onto a gel of agarose
- an electric current is applied
- southern blotting is used to transfer DNA onto membrane
- the presence of DNA is shown using a fluorescent tag
How can DNA profiles be compared?
- DNA profiles can be compared by comparing the total number of bands
- comparing the position of the bands
- comparing the size/ width of the bands
