Lecture One : Hybridization and DNA cloning techniques ( not finished) Flashcards
what’s DNA made of?
phosphate group
deoxyribose sugar
phosphate group
How many strands does DNA have?
2
how many carbons does pentose sugar have?
5
How many hydrogen bonds do each base pairings have?
AT - 2
CG- 3
whats chromatin?
proteins and DNA that are stacked together
minor groove in DNA is _____.
smaller
what are Purine bases?
A and G
what are pyrimidine bases?
T and C
whats a chromosome?
dense packets of DNA by which hereditary information is passed from one generation to the next
whats a autosome?
any chromosome other than a sex chromosom
whats a Gene?
a sequence of DNA nucleotides on a chromosome
whats a Allele?
alternative forms of a gene/marker at a specific genetic location
whats a STR?
short tandem repeat
whats a SNIP?
single nucleotide polymorphism
How do we select the desired target region?
hybridisation
How do you amplify and purify the desired target?
- DNA cloning
2. Polymerase chain reaction (PCR)
what is hybridisation?
two strands of DNA joining together
How can you denature DNA?
- Heating the DNA
2. Exposure to polar chemicals such as formamide or urea
does denaturing DNA do?
breaks it apart into single strand does not damage the DNA
what is Re-forming of the DNA strands known as?
nucleic acid annealing or hybridization.
what can happen when annealing occures?
homoduplexes or heteroduplexes
whats a heteroduplexes?
uplex where base pairing is not perfect across the full
length of a fragment, or when the DNA from each strand is from two different sources.
what can DNA pair with?
DNA, RNA and oligonucleotide sequences.
whats oligonucleotide?
short synthesised, single stranded DNA molecule, ~50 nucleotides long also sometimes referred to as a probe.
oligonucleotide is also known as a ____.
probe
what makes a DNA duplex stable?
bases matching between the two molecules.
when is stability fo a DNA duplex not affected?
short non complimentary regions don’t affect the overall stability
what is stringency?
conditions which will affect weather two strands anneal
what happens when the conditions of stringency are too low ?
conditions too low, probe/DNA will anneal to non complimentary regions
whats the perfect temperature for annealing?
temperature just below the melting temperature (T ) of the DNA or the
oligonucleotide being used.
what happens when temperature is above Tm?
fully based paired hybrids may be unstable.
what happens when temperature below 5°C Tm?
mis-paired hybrids may be stable.
what alters alter hybridization stringency?
temperature and salt concentration
what will high salt concentration do to stringency?
decrease stringency
what will low salt concentration do to stringency?
achieve high-hybridization stringency.
what are the two categories of Hybridization assays?
standard assay, reverse assay
whats a standard assay?
Labeled probe and unlabeled test sample bound to a solid support.
what is a reverse assay?
Unlabeled probe bound to a solid support and labeled test sample
whats microarray hybridization?
Uses the reverse assay method, numerous hybridisation reactions happen at the same time.
How is microarray hybridisation done?
- solid support used to create loads of features on a grid
- Each feature/square contains identical unlabeled oligonucleotides (probe)
- A test sample containing heterogeneous labelled DNA (or RNA) fragments.
- After washing the microarray hybridization signal is read using a laser.
what do the results of microarray hybridisation show?
strong hybridization signal would indicate a lot of complementary sequences between the test sample and the probe in that feature (square on the grid).
can microarray hybridisation be done on RNA?
yes
what are the 5 Hybridization methods in Forensic Science?
- SLP/MLP identification of RFLPs using Southern Blot technique
- Quantification of DNA and first forensic tests using reverse/dot blot technique.
- DNA-DNA hybridization in the polymerase chain reaction.
- SNP identification using hybridization
- Microarray hybridization for biological fluid identification
who developed the southern blott technique?
Edwin Southern in 1973
what happens at the start of a southern blott technique? step 1
- DNA is digested with restriction endonucleases
- Fragments separated by electrophoresis
- Target fragments are identified using labelled probe.
what happens to the gel in the southern blott technique? step 2
Gel is treated with alkali, this denatures DNA, its then transferred to a nitrocellulose or nylon membrane.
what is the membrane soaked in? SBT step 3
soaked in a solution containing a labelled probe.
what happens to the labelled probe? SBT step 4
Labelled probe hybridizes to membrane bound DNA fragments, forming heteroduplexes.
what can the label be in SBT?
radioactive or fluorescent
