what is a cDNA library?
name 4 steps
cDNA library: based on RNA content of a cell
steps:
- extract RNA from cells
- cDNA is synthesized
- RNA is degraded
- cDNA is copied (complementary sequence) to make double stranded cDNA
what is dNTP?
what is the difference with ddNTP?
difference for polymerase?
dNTP: deoxyribonucleotide triphhosphates
ddNTP: dideoxyribonucleotide triphosphhates
> this lacks the 3’ hydroxylgroup needed to form a connection with the next nucleotide
>>>DNA polymerase does not discrimiate between dNTP and ddNTP
chain termination method: DNA sequencing
- what are the ingredients?
- how can you determine the sequence?
- how does the output sequence of chain termination method relate to the sequence to be determined?
chain termination method:
- DNA, primer, DNA polymerase, dNTP, ddNTP
- 1) denature DNA through heat
2) attach primer to 5’ end of DNA fragment, and divide fragments equally over 4 vessels
3) add dNTP, ddNTP (one sort per vessel) and DNA polymerase
4) dNTPs and ddNTPs are attached by polymerase, fragments of different length are made
5) use gel electrophoresis to sequence DNA, shorter fragments travel further in the gel to positive charged site, read out sequence by reading out the position of each sequence in gel - the output sequcene is the complementary to the fragment that is sequenced
problem with chain termination method?
solution?
chain termination method is error prone
>> solution: automatic sequencing
when were more advanced sequencing methods first developed?
> changes?
more advanced sequencing methods first developed in the early to mid 2000s
> methods that could reord the DNA sequence while a DNA strand was being synthesized
> sequencing method was able to monitor/identify the incoporation of each nucleotide in the growing DNA chain
how does iterative pyrosequencing work?
> ingredients?
> mechanisms?
iterative pyrosequencing
ingredients: single stranded DNA template and the four normal dNTP’s
> individual dNTP’s are provided sequentially
> if correct dNTP is provided -> nucleotide incorporation
> simultaneously, a pyrophosphate (PPi) group is produces - > light
> light of light using a CCD camera
> incorrect dNTPs are degraded by apyrase
what are genetic maps?
what are physical maps?
genetic maps:
> depict relative positions of loci based on the degree of recombination: this studies the inheritance/assortment of traits by genetic analysis
physical maps:
> show the actual physical distance between loci (in nucleotides). this approach applies techniques of molecular biology
genetic maps: what gene combinations do you expect in the offspring if both genes are on different chr and independenty assorted?
> what if the results differ?
if genes are on different chr and independently assorted, you expect an offspring ratio of 1:1:1:1
> if results differ, this suggests that genes are linked (on the same chr)
how is the genetic distance between 2 genes on a chr. estimated?
the frequency of which linked genes become unlinked (recombination frequencies) can be used to determine the distance between genes on a chr.
> the further apart two genes are on a chr, the higher the probability that crossing over would occur
> thus a higher recombination frequency would be observed
what is the equivalent of 1 centimorgan on a genetic map?
1 cm = 1% recombination frequency
what are 3 limitations of genetic maps?
genetic maps
- need a large number of progeny and/or multiple generations
- best performed in model organisms subject to selective breeding
- crossing over does not occur at random (maps of limited accuracy)
what are 6 steps if the clone contig approach?
clone contig approach
- genome is broken into fragments up to 1.5 mb
- fragments are cloned in a high capacity vectr
- clone contig is built by identifying clones containing overlapping fragments
- fragments are sequenced
- cloned fragments are anchored onto a genetic and/or physical map
- sequence data can be checked by looking for featues known to be present in a particular region
what is chromosome walking?
how does it work?
chromosome walking: a way to build overlapping series of cloned DNA fragments
how: start with first fragment (A1), hybridize this fragment to all other fragments -> it hybridizes with with E7 and F6 (for example)
> use F6 as a probe to hybridize with all other fragments, it hybridizes with A1 and B12
> continue with B12, etc etc “walk along the chromosome”
whose genome was sequenced in the HGP and the Celera project?
HGP: blood (female) and sperm (male) from ~20 donors (mostly from buffalo) was collected
Celera: DNA from different subjects was mixed (incl different races)
what was an exciting finding in 2007 when craig venters genome was sequenced?
human to human variation is higher than was anticipated earlier. rather than being 99.9% procent identical, its more like 99% identical
what are 4 levels of analysis after genomics?
genomics
> transcriptomics (RNA, gene exression)
> proteomics
> metabolomisc (biochemical blueprint)
> psychophysiology, behavior
what is a clone contig?
clone contig:
> linearlly organized series of cloned overlapping DNA fragments that collectively represent chromosomal DNA sequences
what are DNA libraries?
a comprehensive collection of DNA clones
why is cell-based DNA cloning traditionally used for makeing DNA libraries, and not PCR?
it is more suited to clone large DNA fragments
why is DNA randomly fragmented when making DNA libraries?
only a small proportion of the possible cutting sites are cut by restriction enzyme
what is the main difference between genomic DNA and RNA between cells in one organism?
the DNA content should not differ between cells in one organism
> the RNA content however can vary greatly
what is a major limitation of the chain termination method?
chain termination method
> relies on using gel electrophoresis
> makes sequencing large DNA fragments difficult
what realization lead to a breakthrough in human genome sequencing?
that genetics maps do not have to be based on genes
> but also possible using DNA markers
