[FMS] MCG - Molecular & Cell Genetics - Manipulation of DNA Flashcards
what do restriction enzymes function as?
Restriction enzymes function as ‘molecular scissors’ to cut DNA
they recognize specific nucleotide sequences in the DNA and cuts both strands of the sugar-phosphate backbone
leaves sticky ends
what is a palindrome?
sequence reads the same on both strands in 5’-3’ direction
DNA is visualised in gel by adding what?
ethidium bromide which binds DNA and fluoresces when exposed to UV light.
what sequence do restriction enzymes form?
palindrome sequence
what do plasmids contain that allows replication of the bacterial chromsomes independently?
Plasmids contain an origin of replication that allows replication independently of the bacterial chromosome.
This allows high copy numbers of plasmids, 100-200, to be maintained in each bacteria.
what do plasmids contain that allows the selective growth of bacteria that contain plasmids.
Plasmids also contain antibiotic (AB) resistance genes that allows the selective growth of bacteria that contain plasmids.
outline DNA cloning steps
To clone a DNA fragment the fragment and plasmid are cut with the same restriction enzyme.
Both plasmid and fragment will have same complementary cohesive ends
Cut DNA fragments and cut plasmid are mixed together
DNA fragments will anneal to cohesive ends in plasmid
DNA ligase will ligate the DNA fragment and plasmid together to form recombinant DNA molecule
When cloning eukaryotic genes we need to use ….
mRNA because the introns are removed - cant use genomic DNA with introns because its too large
When cloning eukaryotes, you use mRNA instead of genomic DNA because the introns are removed, however you cant clone mRNA so what do you do?
Complementary DNA (cDNA) is a DNA copy of a mRNA, produced using an enzyme called reverse transcriptase.
therefore for eukaryotic cloning, cDNAs are used as the introns are removed and the clone size is smaller
compare the origin, structure, and function of cDNA and genomic DNA
ORIGIN:
cDNA (complementary DNA):
- synthesized from messenger RNA (mRNA) through a process called reverse transcription.
Genomic DNA:
- complete set of genetic material present in a cell or organism.
STRUCTURE:
cDNA:
- lacks introns, only contains the exonic sequences
- smaller in size
Genomic DNA:
- coding and non-coding regions.
- larger in size
FUNCTION:
cDNA =
- PCR (polymerase chain reaction)
- gene cloning.
Genomic DNA=
- development, functioning, growth, and reproduction of an organism.
- template for transcription
- essential for inheritance and the transmission of genetic traits from one generation to the next.
who was DNA sequencing founded by?
Sanger 1980
outline DNA sequencing method
Uses DNA polymerase to copy single stranded DNA
Makes use of ‘dideoxy’ nucleotides which interrupt the ability of DNA polymerase to copy DNA
DNA polymerase cannot continue to extend the chain of nucleotides after incorporation of a dideoxynucleotide
Method known as the dideoxynucleotide chain-termination sequencing
This method can be automated and allows rapid sequencing of large amounts of DNA
what do ddNTPs have that make them differnt from dNTPs that doesnt allow DNA polymerase to function as normal?
DNA sequencing uses dideoxynucleotides (ddNTPs) which do not have a –OH on the 3’ carbon
DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP as no 3’ OH to form phosphodiester bond
what are the 4 types of dNTPs and ddNTPs
remember, they all function the same way where they ALL LACK -OH ON 3RD CARBON so DNA polymerase cant proceed after they’ve been added into the chain
in automated DNA sequencing how can we tell which dNTP and which ddNTP is what?
theyre marked with fluroescent marker and they can be identified by colour and separated on capillary gel
on each ddNTP has a different wavelength
The laser light excites the fluorescent tag on each fragment as it passes
The wavelength of the fluorescence is read as the fragment passes, and the information is recorded.
3 types of sequencing technologies
Original radioactive dideoxy sequencing on gels
Fluorescent capillary dideoxy sequencing
Next generation sequencing (NGS)
what is PCR
an alternative method to make many copies (clones) of specific DNA fragments without need to use cloning vector
3 stages of PCR
denaturation
primer annealing
extension.
^ These are repeated over and over using a thermocycler to amplify the DNA exponentially.
the number of copies of a fragment in PCR is multiplied by how much in each cycle?
doubled in each cycle:
2,4,8,16, 32, 64, 128, 256 etc
so to work out how many copies made at end of PCR you do 2 to the power of how many cycles ie after 7 cycles how many copies made in PCR - youd do 2 to the power of 7 to give you 128 copies
describe the 3 stages of PCR
Denaturation: the reaction is heated to 95°C to denature the DNA into single strands
Primer annealing: the reaction temperature is reduced to 45-68°C to allow primers to hybridize to their complementary sequences in the target DNA
Primer extension: the reactions temperature is raised to 72°C to allow Taq polymerase to synthesize DNA
^ Amplified DNA fragments are separated by gel electrophoresis
what are DNA microarrays
use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample.
describe the process of DNA microarrays
- all the mRNA from a sample is converted to cDNA,
- fluorescently labelled, denatured into single strands and used as probe.
- This probe is applied to the array which contains spots where single stranded DNA from each gene in the genome is laid out in an array.
how is DNA microarray data read?
the labelled cDNA will bind to the spots on the slides that contain complementary sequences
the spots that bind the labelled will then fluoresce under laser light in a piece of equipment known as a chip reader
the location and brightness of the fluorescent spots on the microarray identify the genes expressed in the test sample and their level of expression
