Unit 5 Flashcards
Polymerase chain reaction (PCR)
artificial method of replicating DNA
What does PCR involve?
repeated cycling through high and lower temperatures
What are the 4 steps of PCR?
1) Denaturation
2) Annealing
3) Elongation
4) Steps 1-3 are repeated
Denaturation
DNA is heated to break hydrogen bonds/separate it into single strands
Annealing
DNA primers attach to the 5’ ends of target sequence at low temps
Elongation
Taq DNA polymerase adds nucleotides
What do primers allow?
the selective copying of a DNA region, bind on either side of the target sequence (on opposite strands)
Taq DNA Polymerase
heat tolerant enzyme
Restriction endonucleases
enzymes that cut DNA into fragments based on recognition of certain sequences
What do restriction endonucleases in DNA?
cut sugar-phosphate
What does an restriction endonucleases generate?
sticky or blunt ends
Sticky ends are…
complementary and single stranded
Gel Electrophoresis
A laboratory technique used to separate and isolate protein or DNA fragments based on size
How are fragments in gel electrophoresis made?
by restriction endonucleases
How does gel electrophoresis work (5)?
- Samples of DNA or protein are placed in a porous gel
- Placed in a chamber and covered with a salt solution, conducts electricity
- The chamber has a negative electrode, where DNA is placed, and a positive
- Power supply generates current that moves samples to positive end
- Small samples move faster than large ones through the gel’s small holes
Genomics
the study of an organism’s complete set of genes and their interactions
Human Genome Project (HGP)
an international effort to determine the complete sequence of DNA bases in the human genome
What were the results of the HGP?
- Humans have 21,000 genes in 3.2 billion nucleotide pairs
- Only 1-2% of the DNA codes for proteins (genes)
DNA Sequencing
process by which the nitrogenous base order of a nucleotide sequence is explained
What does DNA sequencing use?
- PCR (with dideoxynucleotides)
- electrophoresis
Dideoxynucleotides (ddNTPs)
nucleotides that lack the 3’-hydroxyl group necessary for forming phosphodiester bonds; terminate replication
What is the difference between regular PCR and PCR for DNA sequencing?
fluorescently labeled dideoxyribonucleotides are included with normal nucleotides in the PCR solutions
What is different about DNA strands coming from PCRs using ddNTPs?
they differ in length
What does the length of a DNA strand indicate?
the position where a ddNTP was incorporated
What does reading the dideoxyribonucleotides from the shortest to longest fragment do?
it gives the base sequence of the original molecule in the 3’ to 5’ direction
How are fluorescently labeled DNA fragments separated?
based on size using gel electrophoresis
What happens as each DNA fragment reaches the end of the gel?
a laser excites its fluorescent base and a camera detects the color and therefore the base
DNA profiling (2)
- the analysis of DNA fragments to determine whether they come from an individual
- compares sequences from noncoding DNA regions that show variation between individuals
Where is DNA profiling used?
paternity and forensic investigations
Why aren’t genes used in DNA profiling?
they are too similar between individuals
short tandem repeats (STRs)
long stretches of DNA in non-coding regions made up of repeating elements
Who founded the technique of DNA sequencing?
Fred Sanger
Why are STRs useful (2)?
- Every person has several locations were STR repeats are present in their DNA (same locations across different people)
- Each person has a unique number of STR repeats at each location in their genome
How can STRs be cut out?
using restriction endonucleases
What about STR fragments help with identification?
their length
What is needed for there to be a match between crime scene and suspect DNA?
DNA fragments of suspects should be a complete match with the DNA fragments from crime scene
What are the steps of DNA profiling (4)?
- Polymerase chain reaction (PCR) is used to amplify a DNA sample collected from an individual
- Restriction endonucleases are used to cut out short tandem repeats from DNA
- Gel electrophoresis separates the fragments of DNA
- DNA fragments are analyzed
How are DNA fragments analyzed for forensic investigations?
DNA of suspect compared to DNA from a crime scene
How are DNA fragments analyzed for paternity investigations?
child’s DNA fragments are compared to its suspected parents
What must be seen for someone to be considered the biological father of a child on a paternity test?
The DNA that is not covered by the mother must be made up for by the father
