Recombinant dna Flashcards
Recombinant DNA Technology
-It involves combining DNA fragments from different sources, which are not usually found together in nature
-Involves the use of Restriction enzymes along with other strategies for DNA manipulation
Clone
Copy of fragment
Copied DNA can be linear or circular
Recombinant DNA
Cloned DNA fragment
Recombinant fragment to study its structure
Endonuclease
Enzyme that cleaves DNA in the middle of a region/sequence
Exonuclease
Enzyme that cleaves at the end of a DNA fragment
Restriction enzymes
Endonucleases cleave DNA at specific sites
DNA ligases
Enzyme that join two cohesive ends of DNA (ATP dependent)
Eg. T4 DNA ligase
Recombinant DNA technology
Used to isolate, replicate, analys genes
Restriction enzymes background
-in the 1950s, scientists first began to notice bacteriophages were only able to grow in certain bacterial host strains
-This was called “host-controlled variation” and was explained to occur due to the presence of restriction enzymes
Res- 1970
-in the 1970, Werner Weber, Hamilton Smith and Daniel Nathan’s shared the Nobel prize in medicine for discovery of Restriction enzymes
-they discovered that bacteria encoded a restriction factor, called Endonucleases R, that prevented bacteriophages from growing certain hosts while working with Simian Virus
- enzymes selectively cut bacteriophages but not bacterial FNA into fragments of specific and consistent length
-Nathan’s realised it would be a useful tool to map SV40 DNA and its genes
-Not long after, more restriction enzymes were discovered and developed into tools for molecular biologists
RE binding to target DNA
- Work by shape-to-shape matching
-DNA sequence with a shape that matches a part of the enzyme= recognition site
-Wraps around the DNA and cleaves both strands of the DNA molecule
-Each Restriction enzyme binds to unique recognition site: 4-8 bo in length
-Restriction enzyme sequences are mostly palindromic eg: AGCT, GGATCC
What is a palindrome
-Symmetrical sequence that reads the same on both strands in the 5’ to 3’ direction
-2-fold rotational symmetry
-Can form cruciform structures to which proteins such as restriction enzymes can bind
Target DNA structure for RE sites
-Both sides of the DNA strand are cleaved at the same position within the restriction site in each DNA strand
-This can leave either blunt ends (double stranded DNA at the end) or sticky ends (cohesive overhangs)
How RE recognition sites work
No. Of restriction enzymes discovered
-3000 restriction enzymes that recognize over 230 DNA sequences
Type 1 Re
Cleaves DNA at random sites far from its recognition sequence
Type II
Cleaves DNA at defined positions close to or within its recognition sequence
Type IIG
Cleaves outside its recognition sequence with both REase and MTase enzymatic activities in the same protein
Type IIP
Cleaves symmetrical targets and cleavage sites
Type IIS
Recognizes asymmetric sequences
Type III
cleaves outside its recognition sequence and require two sequences in opposite orientation Ms within the same DNA
Type IV
Cleaves modified (eg., methylated) DNA
How are RS are utilized
-Re are used in multiple techniques such as genome mapping by restriction fragment length polymorphisms (RFLPs) and Molecular cloning
Restriction Fragment Length Polymorphisms
-DNA variation- different sources- polymorphisms
-Sources of variation- single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), eg. Microsatellites
-restriction enzymes used to differentiate- differences in length of fragments on the gel
-DNA fingerprinting- genome digested and patterned on gel
-Regions probed for lengths- Southern blotting
-PCR-RFLP-single fragment/region isolated then digested
RFLP genotyping
DNA Fingerprinting
-SNPs may result in the gain or loss of a restriction site, this changes the DNA fingerprint pattern which is called a restriction fragment length polymorphism (RFLP)
-Variations in micro satellite sequence lengths
-RFLP patterns can therefore be used to differentiate between DNA from different individuals, which is useful in forensics
-RFLPs can also be used to determine if DNA belongs to very closely related individuals and offers a useful tool for paternity testing
-Genomic DNA is treated with a restriction enzyme and resolved on an agarose gel
-Fragments are then proved with radioactive markers and viewed. Modern day probes include fluorescent markers
-the resulting pattern of fragments is called a DNA fingerprint
Multi-Locus probes for RFLP
-More information obtained when using multiple probes or probes that bind multiple loci
-Homozygote (AA)- one 5kb band
-Heterozygote (Ab)- three bands- 5kb, 2kb and 3kb
-Both alleles present in extracted DNA
-Band intensity will differ when more DNA is present
STR/VNTR influence on RFLP
-RFLPs can also arise from larger/smaller regions of repetitive DNA
-Different Individuals may have different lengths of repetitive DNA sequences
-Re binding sites on either side of these repeat regions result in different lengths of probed regions
PCR RFLP
-The polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA
-PCR can be combined with RFLP analysis in order to look at a specific RFLP marker
-The gene is first amplified by PCR and then digested with the restriction enzyme
-Digest products are resolved on an agarose gel, and the genotypes of each individual may then be interpreted
Restriction Mapping
-Restriction mapping is a technique used to identify the position of restriction sites on a cloned fragment of DNA
-The DNA clone can either be linear fragment, cloned by PCR or a circular fragment, such as a plasmid, that can be cloned in a bacterial host, such as E.coli
Restriction map
-Establishes a number of, order of, and distances between restriction enzyme cleavage sites on cloned DNA segments
-Created by cutting DNA with different Re and separating DNA fragments by gel electrophoresis, which separates fragments by size
SNPs
Single nucleotide polymorphisms
VNTR
Variable number tandem repeats
-longer than STR
-Also used to identify individuals
- str is less than 25 base pairs while vntr go up to 300 base pairs
-found in promotor region
RFLP
Restriction fragment length polymorphisms
Satellite DNA
-Loci in DNA that have repeating sequences
-Includes STRS and VNTRs
-Is detected by restriction digest
-Areas have high degree of polymorphism where number of repeated is specific to the individual
