DNA Technology and Genomics Flashcards
Why do we clone DNA?
Producing large amounts of DNA of a specific Gene
- Cloned genes provide enough DNA for DNA sequencing. The sequences of a gene can help us understand how a gene works and identify mutations that cause diseases
- Cloned DNA can be used as a probe to identify the same gene or similar genes in other organisms
Expressing the cloned gene to produce the encoded protein
- Large amounts of the protein can be purified to study its structure and function
- Cloned genes can be introduced into bacteria or livestock to make pharmaceutical products such as insulin
- cloned genes can be introduced into plants and animals to alter their traits
- cloned genes can be used to treat diseases – a clinical approach called gene therapy
how do we clone DNA? What is the role of vectors and viruses? How is the DNA cut and inserted into the vector? How can researchers be sure that the vector contains the segment of DNA and that the vector was incorporated into the bacterial cell?
Scientists use bacterial cells as mini-factories to make copies of specific genes.
Goal for recombinant vector to be taken up by the bacteria
Some will take up a single plasmid
Most cells fail to take up a plasmid
Vector carries a selectable maker
How is a DNA library made? what is the difference between a genomic DNA library and a cDNA library?
DNA Library
-Treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments
-DNA Library – collection of many recombinant vectors each with a fragment of chromosomal DNA
-2 Types of common DNA libraries
-Genomic – inserts derived from chromosomal DNA
-cDNA – use reverse transcriptase to make DNA from mRNA of interest (complementary
DNA) – lacks introns so simpler to use
How is DNA amplified without the use of a vector (PCR), describe the steps in PCR, why use heat-resistant Taq polymerase?
Polymerase Chain Reaction (PCR)
- Copy DNA without vectors and host cells
- Goal to make many copies of DNA in a defined region
- Uses high concentration of two primers that are complementary to sequences at the ends of the DNA region to be amplified, deoxynucleoside triphosphates (dNTPs), and a heat-stable form of DNA polymerase called Taq polymerase
- Sample of DNA taken through repeated cycles of denaturation annealing and synthesis
- Themocycler automates this process
- after 30 cycles of amplification, a DNA sample with increase 2^30 fold
First, the temperature is raised to near boiling, causing the double-stranded DNA to separate, or denature, into single strands.
When the temperature is decreased, short DNA sequences known as primers bind, or anneal, to complementary matches on the target DNA sequence. The primers bracket the target sequence to be copied.
At a slightly higher temperature, the enzyme Taq polymerase, shown here in blue, binds to the primed sequences and adds nucleotides to extend the second strand. This completes the first cycle.
In subsequent cycles, the process of denaturing, annealing and extending are repeated to make additional DNA copies. After three cycles, the target sequence defined by the primers begins to accumulate. After 30 cycles, as many as a billion copies of the target sequence are produced from a single starting molecule
How is DNA sequenced?
Determines base sequence of DNA, Sanger Method
- Didoezy chain termination method or dideoxy sequencing
- dideoxynucleoside triphosphates (ddNTP’s) are missing the 3’ – OH group and will terminate the chain
- 4 tubes with many copies of single stranded DNA of interest
- Each tube has a different radiolabbled dNTP
- DNA polyamerase will make complemtary strand until dNTP inserted and chain terminates
- After electrophoresis, DNA sequence can be read by reading which base is at the end of the DNA strand
Sanger Sequencing
- DNA must first be obtained in single-stranded form
- A mixture containing the single stranded DNA, DNA polymerase, the four deoxyribonucleotides, A,T,G,and C, and a single short primer is prepared (primer has a nucleotide sequence that is complementary to the 3’ end of the region to be copied and is required so that DNA polymerase can initiate DNA replication)
- Equal amounts of this reaction mixture are placed in each of four tubes and a different dideoxyribonucleotide is added to each tube
- When a deoxyribonucleotide is inserted into a growing chain, replication continues
- However when a dideoxyribonucleotide is inserted strand synthesis is terminated
- The contents of the reaction tube are then transferred to four lanes of an electrophoresis gel and then the oligonucleotides are seperated by size and nucleotide type
- The shortest oligonucleotide moves furthest down the gel. Reading from bottom to top one base at a time, provides the correct DNA sequence
What are microarrays and why are they useful?
A microarray can identify which genes are transcribed by a cell
- used to monitor the expression of thousands of genes simultaneously
- short sequences of known genes attached to spots on the slide
- goal to find out which genes are transcribed into mRNA in particular sample of cells
- mRNA isolated from those cells and used to make fluorescently labeled cDNA
- cDNAs that are complementary to the DNAs in the microarray will hybridize
what is DNA fingerprinting? Be able to understand the relevance of the bands produced.
- Identifies and distinguishes among individuals based on variations in their DNA
- Chromosomal DNA produces series of bands on a gel
- unique patterns of bands used
- automated using PCR to amplify short tandem repeat sequences
- such tandem repeat sequences are found at specific locations in the genomes of all species, and the number of repeats at each spot tends to vary from one individual to the next
Uses
- Identify different species of bacteria and fungi
- Forensics – 1986 first use in US court system
- Paternity testing and other family relationships
How can we clone a mammal?
- Plants can be cloned from somatic cells
- Believed for decades that mammalian somatic cells could not be used for cloning
- Dolly the first clone
