Manipulating Genomes Flashcards
What is a genome?
The entire genetic material that an organism contains
What are genes?
Sections of DNA on chromosomes
What are introns?
Non-coding sections of DNA
What are tandem repeats?
Repetitive sequences of DNA
Where are tandem repeats found?
Within introns
What is a variable number tandem repeat (VNTR)?
Short nucleotide sequences, organised as a tandem repeat
What are short tandem repeats?
Smaller repeated nucleotide sequences
Describe the method for DNA profiling
- Extract DNA
- Purify with protease to remove histones
- Cut into fragments with restriction enzymes
- Gel electrophoresis
- Nylon membrane placed on gel draws top strand of DNA onto nylon - southern blotting
- Strands fixed in place by UV/heat
- DNA probes added match intron base sequence = hybridisation
Why do restriction enzymes cut DNA at specific base sequences?
- Shape of enzyme active site complementary to shape of a particular sequence of bases in DNA
- ESC can form
- Relevant bonds can be broken (H bonds between bases and phosphodiester from backbone)
How does gel electrophoresis work?
- DNA fragments put into a well in a block of gel
- Along with loading dye (DNA visible)
- Placed in alkaline buffer solution (regulates pH)
- Electric current passed through gel
- DNA fragments move towards positive end (phosphate groups = - charge)
- The smaller the fragments, the further it moves
What is southern blotting?
Designed to locate a particular sequence of DNA within a complex mixture
What are DNA probes?
Single-stranded DNA used to detect presence of complementary nucleic acid sequences by hybridisation
What is hybridisation?
Single-stranded DNA/RNA molecules join to complementary DNA/RNA
Usually, how many different STR’s are looked at when doing a DNA profile?
At least 12
Why are at least 12 different STR’s looked at when doing a DNA profile?
- There is a slight chance that 2 people could have the same pattern for one particular STR in their DNA
- Chances of have the same pattern for 12 different STR’s is much lower
What is a polymerase chain reaction?
A method to amplify a DNA sample by making multiple copies of it for analysis
What “ingredients” are needed for PCR?
- Original tiny DNA sample
- Excess of free nucleotides
- Primers
- DNA Taq polymerase enzyme
What are primers?
Short pieces of single-stranded DNA which start the copying process
Describe the steps in PCR
1- Denaturing phase
- temp inside machine = 90°-95°C for 30 secs
- breaks H bonds between strands
2- Annealing phase
- temp decreased to 55°-60°C
- primers join to both ends of single DNA strands
3- Extension phase
- temp increased to 70°-75°C for 1 min
- optimum temp for DNA Taq polymerase to work
- adds bases to primers, extending complementary strands
What type of DNA polymerase is used in PCR?
Taq polymerase that isn’t denatured by high temps
Why might you not get as many copies of the DNA made as expected?
- May not be enough primers
- Primers may not attach to all DNA strands
- Insufficient nucleotides available
Give 3 uses of DNA profiling
1- Criminal investigations
2- Prove paternity of a child
3- Identify individuals at risk of disease
What is DNA sequencing?
The process of determining the nucleic acid sequence