Genome Flashcards
The genome
ALL the DNA in a cell/organism
To sequence a genome
Work out the entire DNA base sequence of a cell/organism
Why can we work out amino acid sequence of proteins from the sequenced genome?
For each gene, we can work out AA sequence from every triplet
Easy because no introns means the whole genome will encode for AA sequence in polypeptide
Proteome
The full range of DIFFERENT proteins that can be produced from a CELL
Why is it harder to work out translation of genome to proteome in eukaryotes?
Presence of introns, non coding base sequences need to be removed before predicting AA sequence of polypeptide
Promoter needs to be identified = show start of gene
Why is it hard to work out which proteins will be produced from each eukaryote cell?
Because different cells have different transcription factors specific to promoter so hard to identify promoter thus start of gene
What do gene technologies do?
Study of gene function and alteration using various technological processes
List of gene technologies
Recombinant DNA technology
Restriction endonucleases
Gene machine
Amplification of DNA fragments - PCR
Main idea behind recombinant DNA technology
Transfer of DNA fragments from one organism
To another (the transgenic organism) which translates for a polypeptide within cells
Why is recombinant DNA technology possible
Genetic code is universal so same DNA base sequence encode for same AA sequence thus same polypeptide IN ALL SPECIES
Transcription and translation mechanisms are the same IN ALL SPECIES
Main use of recombinant DNA technology
Take human gene eg for insulin
Put in bacteria
Mass produces protein by translation so can be harvested for medical treatment
How can we produce the fragments of DNA in the first place for recombinant DNA technology?
Reverse transcriptase
Restriction endonucleases
Gene machine
Using reverse transcriptase to produce fragments of DNA (to be transferred to new organism)
Catalyses formation of complementary DNA from an mRNA template in reverse transcription
How to obtain reverse transcriptase
Collect from virus eg HIV
Purify to obtain reverse transcriptase on ITS OWN
Why would we use reverse transcriptase to produce DNA fragments?
Non coding base sequence in DNA in eukaryote genes
Need to splice introns out from pre-mRNA before the mRNA is translated
Prokaryotes lack the ability to splice them out of pre-mRNA
and if eukaryote gene is given to prokaryote, then introns need to be spliced out, this method makes it work
AND A LOT OF MRNA PRODUCED SO MORE USED THAN GENE ITSELF
Using reverse transcriptase to obtain DNA fragment method
pre mRNA complementary to eukaryote gene is produced a lot in a cell then is SPLICED so only has coding bases
By complementary base pairings, single strand of DNA is formed catalysed by reverse transcriptase
Isolate single strand and form double strand using DNA nucleotides catalysed by DNA polymerase = copy of DNA gene WITH NO INTRONS
cDNA
Single strand of DNA formed in reverse transcription formed from condensation reaction of DNA nucleotides catalysed by reverse transcriptase
Complementary by base pairs to spliced mRNA
How is cDNA isolated
Hydrolysis of mRNA template strand using specific enzyme with active site complementary to mRNA ONLY
What enzyme catalyses formation of second DNA strand from cDNA?
DNA polymerase
Bonds formed between DNA nucleotides?
Phosphodiester bonds
Restriction endonuclease method
Cut a specific sequence of bases in DNA (gene) out from a molecule to obtain DNA fragment
Catalysed by enzymes called reverse endonucleases
How do reverse endonucleases work?
Reverse endonucleases Has a shape of active site that is complementary to the shape of a specific DNA base sequence
Therefore each enzyme cuts DNA at a specific base sequence
Gene machine general outline
Making of any DNA base sequence from scratch
By working out AA of desired protein then working backwards to get the base sequence of a gene = intron free
as oligonucleotides
Oligonucleotides
= free nucleotides to make lots of short single stranded DNA in a gene machine
What is done with the oligonucleotides from gene
Overlapped and made double stranded DNA by PCR then amplified to make more copies of gene
Why is gene machine not ethical?
May involve making of a gene that doesn’t exist naturally
Amplifying DNA fragments 2 methods
In vitro (out of body)
In vivo (in body)
DNA amplification
Making lots of copies of a specific DNA base sequence
So mass produce a lot of polypeptides
Gene clothing
Specific genes having identical copies amplified in PCR
