Recombinant DNA And Biotechnology 11-16 Flashcards
DNA from a cDNA library can be cloned into an ________ _______ for _______ of ___________
Expression vector
Production of proteins
Bacterial expression vector
-promoter, shine dalgarno sequence and cDNA are used to transform expression bacteria strains
Why use yeast as a vector?
Bacteria have no organelles
Yeast can express cell surface proteins
DNA sequencing is a technique used to determine…
The exact sequence of a cloned or PCR amplified stretch of DNA
How do you get a single stranded DNA template?
Melt it
What are major components of the reaction used to conduct sequencing
DNA + dNTPs + DNA primer + polymerase
What does each of the 4 tubes contain in DNA sequencing
- ddCTP
- ddATP
- ddGTP
- ddTTP
What stops elongation of DNA strand?
DdNTP
-used in cancer treatments
What are probes used for
Identify DNA fragments
What is a probe
A ssDNA molecule, labeled using radioactivity that be by hybridized a ssDNA that is complementary
Hybridization
- DNA made single stranded
- target immobilized on support so it wont reanneal
- ssDNA coated membrane exposed to probe
- if complentary seq Preston, probe will bind and can be identified via autoradiography
Smaller probes
- chemically synthesized oligonucleotides (2-30 base pairs)
- very specific
- 1 nucleotide difference can affect recognition
- DIAGNOSTIC
Larger probes
- made via one of several molecular biology techniques
- much less specific
- heritage
- COMPARATIVE
Which type of probe is diagnostic
Smaller
Which type of probe is comparative
Larger
Southern blotting
- analysis of DNA
- restriction digestion
- gel electrophoresis
- denatured and blotted
- blot probed
Northern blot
- analysis of RNA (mRNA)
- complentary mRNA
- only detects expressed sequences
- can be used for tissue or cell specific studies
- QUANTITATIVE (to measure gene expression)
Western blot
- analysis of protein
- probe is antibody specific to the protein of interest
- quantitative
What is the frequency of nucleotides that are different in humans who are not related
1 in 1500
Genetic variations in non-coding regions with no disease
Polymorphisms
The term ______ is usually reserved for genetic changes that cause disease
Mutation
When is a RFLP present?
If a genetic change in a polymorphic region creates or deleted a restriction site OR has more or less of a repeated sequence
DNA variations resulting in RFLPS
- Single nucleotide polymorphisms (SNPs)
- variable number or tandem repeats (VNTRs)
Single nucleotide polymorphisms (SNPs)
- single nucleotide change
- 90% of genetic variation
- may create or abolish restriction site***
- occasionally an RFLP is created by a disease causing mutation
- more often it is a harmless change that results in a different restriction pattern
Variable number of tandem repeats (VNTRs)
- human genome contains many regions where a sequence is repeated in tandem many times
- varies from person to person and unique for individual
- if DNA is cleaved on either side of a VNTR, an RFLP is produced
RFLPs based on SNPs
- creation of abolishment of a restriction site
- different size RFLPs based on different restriction cutting
- used to mark genes (alleles), disease markers
RFLPs based on VNTRs
- more or less of a tandem repeat
- different size RFLPs based on different number of repeats
- used as molecular fingerprints (paternity tests)
DNA cloning
Amplify fragments by inserting them into a vector and the host replicating the vector
PCR
Amplification done completely in a tube
