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Genetics Exam 3

Terms in this set (37)

Recombinant DNA
DNA resulting from combining DNA from two sources
Sources of Recombinant DNA
- Crossing over during meiosis to increase genetic diversity
- Chromosomal reaarangements which drive evolution
- Transposons and some viruses
- Development of immune response cells
- modern molecular techniques
Cloning
- making genetically identical copies of DNA molecules, cells, or whole organisms
Genetic engineering
Using the processes of cloning and recombinant DNA to mindfully modify a gene, cell, or organism
1971
birth of modern biotechnology
1978
recombinant human insulin
2011
94% of US soybean, 75% US cotton, greater than 73% US corn is transgenic
Basics for making recombinant DNA
Plamids = vectors to carry rDNA
Restriction endonucleases = to cut DNA
DNA ligase = to connect DNA
E.coli = used to amplify cloned DNA
Plasmids
Bacterial extra-chromosomal DNA
Circular DNA molecules
- Stringent = 1-2 copies per cell
- Relaxed = usually over 100 copies per cell
Exchange genetic material within or between species
Restriction Endonucleases (RE)
Cut DNA at specific sites on the DNA
Endonuclease - cleaves DNA internally
Restriction -from its natural function - restricts virus infection in bacteria
Bacterial Defense
Cleaves at specific sequences of the phage DNA but not the bacterial chromosome because of methylation.
Palindromic
A sequence that can be read the same forwards as backwards (5' and 3'), RE cleaves the phospodiester bond here.
Types of RE
4,5,6,8 base pair recognition for cleaving
smaller recognition sites produce smaller DNA fragment
Ends of RE
blunt ends AluI and BalI
5' overhangs - cohesive ends EcoRI and HindIII
3'overhangs - cohesive ends
DNA Ligase
Connects newly synthesized DNA fragments
DNA ligase + ATP is used to form new phospohodiester bonds between fragments that are covalently linked
Non-proofreading DNA polymerase
Taq Pol can either add an A overhand or a G overhang during PCR.
In-Fusion cloning
Gene-specific PCR product + Any linearized vector = any linearized vector = recombinant vector.
Direct PCR cloning into any vector you want.
Two method to introduce a plasmid into bacteria
-Treat cells with calcium ions and use brief heat shock
- electroporation = brief high intensity pulse of electricity
Polymerase Chain Reaction (PCR)
Copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities
Oligonucleotide Primers
One complementary to the 3' end of one stand of DNA and another complementary to the 3' end of the other strand.
Critical Components of PCR
1. Template (target) DNA
2. Oligonucleotide primers specific to target regions
3. Nucleotide building blocks
4. taq DNA polymerase
PCR Steps
1. Denaturation (high temperatures)
2. Annealing of Primers (low temperatures)
3. DNA synthesis (intermediate temperature)
Reverse Transcriptase PCR
- generate cDNA from mRNA
- use PCR to copy the single stranded DNA into a double-stranded DNA
Quantitative PCR (qPCR0 and RT-qPCR
Quantifies abosulte number of copies made in real time.
Genomic library
- contains at least one copy of all of all sequences in the genome ( exons, introns, regulatory sequences, intergenic DNA)
Cutting DNA with restriction enzyme with ligating the fragments into vectors
vector depends on the size of the genome.
Genomic equation
N = ln(1 -P)/ln(1-f)

N = the number of required clones
P= probability of recovering a sequence
f = the fraction of the genome in each clone
subgenomic library
- subgenomic libraries, single chromosome generated by methods such as flow sorting and pulsed -field gel electrophoresis
cDNA library
complementary DNA copies made from the mRNA's present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation
Creation of the cDNA library
- isolate mRNA from cells
- synthesize the complementary DNA using reverse transcriptase
- cloning the cDNA molecules into a vector
Probes
Complementary to part of the gene
DNA fragments labeled either with a radioisotope for autoradiography or an enzyme for colorimetric detection.
cont..
Clones in a library are cloned on a plate to produce colonies
- colonies transferred from plate to a filter, filter hybridized with a nucleic acid probe to the DNA sequence of interest.
Restriction map
- establishes the number and order of restriction sites and the distance between restriction site on a cloned DNA segments.
Southern Blot
- identify which clones in a library contain DNA sequence and to characterize size of fragments.
What do southern blots determine
- determine whether a clone contains all or part of a gene
- ascertain the size and sequence organization of a gene or DNA sequence of interest
Northern Blot
Specific probe for diphenol oxidase
DNA sequencing
The most common method of DNA sequencing: dideoxy chain termination sequencing
Dideoxy Sequencing
- Separate by gel electrophoresis
- use polyacrylamide gels (small pores, seperates better)
- monomer is neurotoxin
- polymer safe
400 -600 fragmentsizes
radioactive labeled (x-rayed)
chart (fluroscent label)
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