Biotechnology Flashcards
Polymerase chain reaction (PCR)
amplify specific DNA sequence
- require very little target sample
- many uses:
- medical diagnosis, identify organisms,
cloning, DNA sequencing, gene expression,
detection of mutations, forensics, etc.
Recombinant DNA technology
use of a combination of techniques to
clone and manipulate genes for study or
applications
- restriction endonuclease cleavage of gene
- cut DNA into manageable & precise fragments
- cloning into appropriate vector
- polymerase chain reaction (PCR)
amplification
- Southern, northern, western blotting
PCR
1) Denaturation- DNA template DNA primers, dNTPs are heated to separate the DNA strands
2) Annealing- sample is cooled DNA primers anneal to specific sequence to be amplified on the DNA template.
3) Elongation- temperature is increased to 72 C. DNA polymerase adds dNTPs to the strand to replicate the sequence after each prier.
CRISPR/Cas9
Genome editing tool derived from bacteria. Consist of RNA (gRNA) 1) which is complementary to target DNA sequence and an endonuclease (Cas9) which makes a single or double strand break at the target site. 2) imperfect segments are repaired by non homologous end joining (NHEJ) accidental framshift mutations (knockout). IE., removing virulene factors from pathogens, replacing disease causing alleles of genes with healthy variants (in clinical trials for sickle cell disease, and specifically targeting tumor cells.
Recombinant DNA technology (Vectors)
vehicle for carrying DNA of interest - able to expand number of copies of genes - for other uses, eg. sequencing, transfer to other expression vectors (yeasts, eukaryotes, viruses) - bacterial (ie. plasmids), viruses, yeasts, etc. - chosen depending on size and function - can be used to produce proteins - eg. insulin, growth factors, vaccines, etc. - expression vectors - driven by proper transcription promoters - applications in gene therapies
Recombinant DNA- DNA libraries
DNA libraries - collection of cloned DNA fragments • Genomic library - every complete DNA sequence of the organism - coding and noncoding sequences, transcription regulatory sequences, introns, etc. - restriction digest of entire (human) DNA and insert every fragment into vector
Recombinant DNA- Complementary DNA (cDNA) library
Complementary DNA (cDNA) library - protein coding sequences, ie. mRNA - mRNA is converted to DNA by reverse transcriptase - do not contain introns or regulatory sequences
Southern blot hybridization (DNA)
detects specific DNA sequences
uses gel electrophoresis to separate DNA fragments
- transfer to membrane support
- probe (hybridize) with a specific DNA sequence
- bind to complementary DNA sequence on membrane
- use to measure gene copies, detect mutations, DNA modifications (eg. DNA methylation) identification by restriction fragment length polymorphism (RFLP, also DNA fingerprinting) - used to detect mutations and polymorphisms
Northern blot hybridization (RNA)
detecting presence of specific RNA sequences
- determine the lengths of RNA sequences
- measure transcription levels (ie. mRNA) of genes
- alternative splicing of mRNA
- detect splicing errors
- summary: - similar to Southern blot, except use RNA - probe with specific DNA sequence
Western Blot (Proteins)
detecting presence of specific proteins
- separate total proteins in gel
- transfer to membrane
- probe with antibodies
- specific to protein of interest
- also detect modifications (phosphorylations, ubiquitinations)
Restriction fragment length polymorphism (RFLP) analysis of DNA sequences utilizes restriction enzymes to cleave DNA prior to gel electrophoresis.
Which of the following DNA sequences would be easiest for a restriction enzyme to act upon?
AGGCCT- PCR uses restriction enzymes to cut out the specific sequence of DNA that is going to be amplified. These restriction enzymes look for palindromic sequences to cut in the middle. A palindromic sequence is one whose complementary DNA strand is synonymous to its original strand. We always assume the strand is given to us in the 5’-3’ direction unless otherwise indicated. Here, 5’-AGGCCT-3’ & 3’-TCCGGA-5’ : This is the same sequence.
TAKEAWAY- PCR amplification of a DNA strand requires a palindromic sequence in order for restriction endonucleases to cleave DNA strands. A palindromic sequence is one whose complementary strand reads the same to the original strand in the same direction (5’-3’).
A researcher is studying the incidence of the sickle cell trait in African American infants. To identify the trait, polymerase chain reaction testing is performed on venous blood samples obtained from the infants.
Which of the following is required for this laboratory technique?
Initial sequence of the 3’ end of a DNA strand. This is the correct answer. The polymerase chain reaction (PCR) test detects and multiplies a specific target segment of a DNA strand using primers and the enzyme Taq DNA polymerase. The first step in PCR is heating the test sample (venous blood, in this question) to denature the double stranded DNA into single strands (denaturation step). Primers, which are composed of nucleotides that are complementary to the 3’ end of the target DNA sequence nucleotides, binds to the 3’ end of the target DNA –known as the hybridization step. This then coverts the single-stranded DNA segment into a double-stranded structure –allowing the enzyme, DNA polymerase to make new complementary DNA strands, identified as the amplification step.
TAKEAWAY: Polymerase chain reaction (PCR) is a useful diagnostic tool that detects and amplifies a specific region of interest on a DNA strand. It follows a process that begins with denaturation, following annealing and ends with elongation. Heating and cooling cycles are continued until the DNA sample size is sufficient. With that being said, primers –during the annealing step – anneal (hybridize) to the initial sequence of the 3’ end on the DNA strand of interest to aid in the amplification process.
When comparing DNA replication in prokaryotes and eukaryotes, it is demonstrated that a prokaryotic model such as E. coli can replicate in approximately 40 minutes and a eukaryotic genome, while many orders of magnitude larger, can replicate within just a few hours.
Which of the following characteristics of eukaryotic DNA replication best describes these findings?
Simultaneous replication at multiple origins. This is the correct answer. DNA replication is initiated at a specific sequence within DNA identified as the origin of replication. While the eukaryotic genome has multiple origins of replication, there’s only one fixed origin in prokaryotes (where specific proteins that initiate replication can then bind to).
TAKEAWAY: The rate of replication in mammalian cells are exponentially higher than in E. coli. A much higher number of helicases is required for eukaryotic cell replication than for prokaryotic cell replication. The human genome has many starting points for replication known the origin of replication, making it a much faster process in replication.
A team of geneticists and biomedical engineers have created a ground-breaking new technology to watch eukaryotic DNA replication in real time. As they record the enzymes and other structural proteins working together, they take note of the polymerases that are capable of 3’ to 5’ exonuclease activity.
Which of the following eukaryotic polymerases is not capable of this process?
Polymerase alpha – This is the correct answer. Known for being the equivalent to the prokaryotic primase, this polymerase is incapable of proofreading. 3’ 5’ exonuclease activity allows for polymerases to proofread their work on a growing DNA strand. In other words, if a polymerase identifies an incorrectly paired nucleotide, it can immediately remove and replace that particular nucleotide.
TAKEAWAY: Of the eukaryotic DNA polymerases, alpha and beta are incapable of 3’ 5’ exonuclease activity, or proofreading. Polymerase beta, although not mentioned in the question is utilized by the cell for repair primarily. Polymerases gamma, delta and epsilon are all capable of proofreading.
Polymerase delta
Aids in the elongation of Okazaki fragments of the lagging strand. It does have the ability of proofreading.
