39 - Recombinant DNA technology Flashcards
Manipulation of a DNA sequence and the construction of chimeric molecules
Genetic Engineering
The combination of genetic material of 2 or more organisms
i.e. Human DNA + bacterial DNA
Chimeric Molecules
DNA Review:
What are exons?
- Coding region
- remains in the mature mRNA after processing of primary transcript
- shorter sequences
DNA Review:
What are introns?
- non-coding region
- are the ones removed during the processing of the primary transcript
- in between exons
- not present in mRNA
- longer sequences
(True/False)
Introns are always present in mature mRNA
False
During processing, introns are removed and exons are the ones that compose the mature mRNA
The process of removing the introns in the primary transcript before it is transported to the cytoplasm
RNA splicing
What is the 5’ Flanking Sequence DNA?
- found before the transcription start site
- contains regulatory elements (i.e. promoters, enhancers, silencer)
- determine WHEN and HOW MUCH primary transcript are produced > Gene Expression
The region that precedes the starting point of a gene, also known as the transcription start site. This sequence is typically located on the 5’ end of the DNA strand.
5’ flanking sequence DNA
Definition:
Promoter regions
Specific sequences of DNA that are recognized by RNA polymerase, the enzyme that synthesizes the mRNA transcript.
Definition:
Enhancers
Regulatory elements that can increase the transcriptional activity of the promoter.
Definition:
Silencers
Regulatory elements that decrease transcriptional activity.
Sequences of DNA that control the transcription of a gene by determining when and how much mRNA is produced.
Regulatory elements
The process by which the expression of a gene is controlled, typically through the activity of regulatory elements and proteins.
Gene Regulation
Review:
DNA Structure
Review:
DNA Structure -Bonds
Review:
Nucleic Acid Structure
Type of DNA preparation where mRNA as the sample is converted to DNA
Enzyme reverse transcriptase is used
copy or complimentary DNA
cDNA
Type of DNA preparation that digest extracted DNA with a restriction enzyme
Genomic DNA
A restriction enzyme that cut the DNA at specific DNA sequence within the molecule
Endonucleases
A restriction enzyme that digest from the ends of DNA molecule
Exonucleases
Enumerate the types of restriction enzymes
- endonuclease
- exonuclease
A single-stranded DNA molecule used to detect complementary DNA sequences.
Can be labeled with detectable markers for visualization and quantification
DNA probe
Hybridization is used to bind the probe to a sample containing the complementary sequence.
The probe and target DNA form a stable hybrid that can be detected and quantified
From DNA it makes complimentary DNA copies
Used in:
1) creating DNA probes
2) DNA amplification
3) DNA Sequencing
DNA-dependent Polymerase
Derived from RNA tumor viruses that make cDNA copies of RNA templates
Useful in cloning sequences that are complimentary to mRNA
RNA-dependent polymerase
Useful in cloning sequences that are complimentary to mRNA
Restriction enzyme origin
Found in Bacterial genes
used to protect short bacterial DNA from infective phages that try to attach themselves to the DNA for proliferation
An organism with a restrictive enzyme will always have
site specific DNA methylases
Restrictive enzymes cut DNA at specific sequences. If a bacteria has a restrictive enzyme for XXX sequence to protect from viruses with XXX sequence, the bacteria will have DNA methylases to protect its own XXX sequence from its own restrictive enzyme
Site specific DNA methylates
Enzymes that methylates host DNA
Sticky ends
specific DNA sequence that forms after DNA is digested by restrictive enzymes
useful in constructing hybrid or chimeric DNA molecules
Characteristic linear array of sites for various enzymes
Restriction map
- Result from cutting both strands of DNA in the same position
- No overhanging nucleotides (homopolymer tailing)
- Fragments cannot base pair with other blunt ends
- Difficult to join together without additional enzymes or techniques use enzyme: terminal transferase
Blunt ends
- Result from cutting DNA in a staggered fashion
- Overhanging single-stranded DNA sequences at each end
- Fragments have complementary overhangs that can base pair with each other
- Can be easily joined together using DNA ligase
- Commonly used in recombinant DNA technology
Sticky ends
Formed by endonucleases that cleaves a specific double-stranded DNA sequence (4-7 bp long)
Seals overhangs to make recombinant DNA by catalyzing the formation of phosphodiester bond between adjacent 3’-hydroxyl and 5’ -phosphate ends of DNA
DNA ligase
Carrier of target DNA
Vector
Essential features of Vectors
- able to replicate
- capable of insertion of DNA of interest
- selectable marker
- contain a site for insertion of target DNA
Extrachromosomal circular pieces of DNA found in bacteria
Can replicate within the host bacterium
Plasmids
Caryy genes that code for proteins that confer antibiotic resistance to the host
Type of vector (?)
Small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance
Plasmids
Viruses that infect bacteria
can be genetically modified to produce cloning vectors
- more efficient in transecting the host
- can hold a bigger fragment of DNA
- linar DNA
Bacteriophages
Plasmids that contain DNA sequences (cos sites) required for packaging lambda DNA into package particles
Is a DNA cloning vector that combines the best features of plasmids and phages
cosmid
Accept 1000kb inserts and are easily propagated in yeast cells
Helpful in studying Human genome
YAC or Yeast Artifical Chromosome
Vectors SUMMARY
Identical host cells that carry an identical recombinant DNA molecule
Clones
Basic cloning strategy
1) Recombinant DNA is made (from vector and target DNA by annealing and ligation)
2) Recombinant DNA is introduced to the host via transformation or transfection
3) propagation in host bacterial cells
4) target DNA is specifically selected by hybridization
Gene cloning steps
1) vector and insert DNA are digested with the same restricion enzyme (ends of insert DNA and vector DNA match)
2) restriction digests of vector and insert DNA are mixed and allowed to reanneal with DNA ligase
3) vecrtor is propagated in bacteria
4) target DNA selected by various detection procedure
Blotting Techniques
“SNoW DRoP”
Southern Blotting - DNA
Northern Blotting - RNA
Western Blotting - Protein
Can show overexpression of oncogenes and down regulation of tumor-suppressor genes in cancerous cells
Used in gene expression in the rejection of transplanted organs
Northern Blot
Used to determine whether a transgenic organism expresses the inserted gene to produce a protein
Western Blotting
Used for prenatal diagnosis
Southern Blot
Allows one to observe a particular gene’s expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, etc.
Northern Blot
Uses labelled antibodies to detect proteins
Western Blot
Chemical DNA sequencing by
Maxam & Gilbert
Enzymatical DNA sequencing by
Sanger
(True/False)
The chemical DNA sequencing procedure is easier and quantitatively more superior than the enzymatic procedure
False
Used for DNA amplification
Polymerase Chain Reaction (PCR)
RTPCR
Reverse Transcription Polymerase Chain Reaction
In RT-PCR, RNA molecules are first converted to complementary DNA (cDNA) using the enzyme reverse transcriptase.
The cDNA is then amplified using the polymerase chain reaction (PCR) technique to produce millions of copies of the target RNA sequence.
A variation of RFLP analysis
DNA Fingerprinting
A method for detecting protein-DNA interactions by applying labeled DNA probe to a transfer membrane that contains renatured protein
Southwerstern Blot
Comprises complementary DNA copies of the population of mRNAs in a tissue
cDNA Library
What is a tool in the diagnosis of single base changes like sickle-cell disease?
Restriction Fragment Length Polymorphism
