DNA Technology Flashcards
(19 cards)
Genome
the complete set of genetic information contained within the cells of an organism
Proteome
the full range of proteins that can be coded for by the cell of an organism
Genome Sequencing and Uses
Proteome Sequencing and Uses
Recombinant DNA Technology
The transfer of DNA fragments from one organism to another, possible as the genetic code is universal, mechanisms of transcription and translation are universal
Producing DNA Fragments: Reverse Transcriptase
Insulin Example:
1)mRNA coding for insulin taken from beta cells.
2)Reverse transcriptase used to form single stranded cDNA from mRNA template
3)Double stranded DNA is formed on the template of cDNA using DNA polymerase
Producing DNA Fragments: Restriction Endonucleases
A restriction enzyme cuts the double stranded DNA molecule at its specific recognition sites. This produces DNA fragments with either sticky or blunt ends. DNA ligase can be used to join the DNA fragments together.
Producing DNA Fragments: Gene Machine
Amplifying Fragments: PCR (in Vitro) Process
1) Heat to 95 degrees to break hydrogen bonds to separate DNA strands.
2) Cool to 55 degrees and add DNA primers, DNA primers anneal to complementary bases
3) Heat to 72 degrees and add free nucleotides which will bind to complementary bases and DNA polymerase which forms phosphodiester bonds between adjacent DNA nucleotides
4)Repeat
PCR Uses, Problems, Advantages
Uses: Paternity testing, testing for inherited diseases, forensics
Problems: needs a pure DNA sample, contaminated DNA will also be amplified
Advantages: rapid, doesn’t need living cells
DNA Primers
Short sequences of DNA nucleotide bases that have a set of bases complementary to those at one end of each DNA fragment.
Starting sequence for DNA polymerase to work from
Prevent two strands of DNA fragments from rejoining
Amplifying Fragments: Using Vectors
1) Restriction endonuclease cut DNA fragment with sticky ends from DNA of cell that produces desired proteins
2) Same restriction endonuclease cuts plasmid from a bacterium, also with sticky ends
3) Sticky ends of plasmid and DNA fragment complementary and DNA ligase is used to join them together
4) The bacterial cells mixed with vectors and heat shocked so they are taken up and bacterial cells divide cloning the DNA fragment
Marker Genes
To identify the cells that have taken up the DNA fragment, marker genes are also inserted into vectors.
E.g. Antibiotic resistance, fluorescence using UV light, enzyme producing gene such as lactase
DNA Probes
short single strand of DNA complementary to a target base sequence with an identifiable label, e.g. radioactive or fluorescent label.
They are added to a sample of single stranded DNA to identify particular alleles as they will bind it that allele is present.
Uses of DNA Probes
Screening for inheritable disorders.
Screening for mutated genes such as oncogenes
Personalised medicine, how someone will react to a drug and dosages
DNA Hybridisation
Genetic Fingerprinting
Everyone’s genome contains non coding sections of DNA called VNTRs, which are unique in number and length for every individual. Two people having the same VNTR is very unlikely so it can be used to identify who a DNA sample came from.
Used to identify who was at crime scenes
Genetic Fingerprinting Process
DNA extracted from sample and amplified
Restriction endonucleases cut the DNA into fragments containing just VNTRs
DNA fragments separated using gel electrophoresis
Transferred from the gel to a nylon membrane
Gel immersed in strong alkali to split double stranded fragments into single strands.
DNA probes are added to label the VNTRs
Use X-ray or UV light depending on probes to reveal bands where the DNA probes have attached (the VNTRs)
Gel Electrophoresis
DNA fragments placed on one end of the gel and an electric current is applied along the gel
DNA is negatively charged and so is attracted to the positive end and moves across the gel.
The smaller fragments move faster and so travel further.
So the DNA fragments are separated on the basis of size
