chapter 19 Flashcards
what is rna replication
- use of enzyme to create a complementary copy of rna
what is dna replication
- geneoems dna is copied in cells
what is transcription
copying dna into rna using rna polymerase( main enzyme)
what is translation
genetic information from rna (mRNA) into amino acids
- protein synthesis
what is gene regulation
the process of controlling which genes in a cells DNA are expressed
what is molecular genetic techniques
techniques used to locate,analyze, alter, sequence study and recombine
what are 4 key innovations
- recombinant dna tech
- polymerase chain reaction
- DNA sequencing technology
- genome edititing methods
Define recombinant DNA technology
Set of molecular techniques for locating, isolating,altering and studying dna segments
Define restriction enzyme (restriction endonuclease)
Recognize specific nucleotide sequences in dna and make double stranded cuts at those seqauences(restriction sites)
restriction enzyme is produced naturally by…
bacteria and are used in defense against viruses
how does a bacterium protect its own DNA from restriction enzyme
adding methyl group to the dna
Describe the origin of restriction enzymes and how their names are derived
from bacteria and their name is derived from the name of the restriction enzyme being used
how are restrictin enzyme name derived
- begins with abbrev. of the bacterial origin
- first 3 signify the bacterial enzyme, the 4th letter is the strain
Describe the 2 types of cuts restriction enzymes can make (Fig 19.2a)
- HindIII
-PvuII
what does restriciton enzyme hindIII do
cuts the sugar phosphate backbone of each strand and cuts staggered cuts in dna producing single stranded cohesive ends
what does restriction enzyme pvuII do
PvuII cuts both strands of dna straight across producing blunt ends
when are restriction enzymes used
Restriction enzymes are used whenever dna fragments must be cut or joined
how can restriciton enzyme create recombinant dna
In a restriction reaction dna is placed in a small tube with the buffer solution and a small amount of restriciton enzyme then it is heated and the enzyme will have then cut the appropriate restriction sites
how can gel electrophoresis separate dna fragments by size
uses an electric current to separate different sized molecules of DNA in a porous sponge like matrix
what charge is dna
negative
where does the dna migrate in electrophoresis
toward the positive charged electrode
in gel electrophoresis what moves more quickly through the gel
- shorter strands move more quickly than longer strands so the fragments are arranged in order of size
what is CRISPR-case9
technology that allows scientists and medical researchers to edit parts of the genome by adding or altering DNA sequence
scientists have engineered crRNA from what
bacteria with a sequence that is specific to the target sequence of interest
the crRNA sequence pairs with what
CAS9 protein
what does CAS9 do
cuts dna at a specific location in the genome
steps for CRISPR-cas9 genome editing
- crRNA from bacteria that is specific to the sequence of interest
- other part of crRNA has a sequence that pairs with CAS9 protein
- complex finds and binds to the target DNA sequence
- CAS-9 makes double stranded cuts in the target DNA sequence
- nonhomo ends join ( repair pathway that repairs chromosome breaks)
- donar DNA can be inserted into the cell with ends complementary to the break sequence
explain how CRISPR- cas9 can be used in gene therapy
- can be used to correct disease causing mutation
- silence genes
- prevent or cure disease
- study gene function
what are the different uses of CRISPR-cas 9
- gene therapy
- treating disease
- gene modification
- change gene expression
- detect pathogens
what is a probe
Probe: piece of DNA that is complementary to sequence being searched for– has a label on it, ie radioactive so it can be seen
what is PCR stand for
polymerase chain reaction
what is PCR
laboratory techniqye used to amplify dna sequences
what does replication by pcr require
Replication by PCR requires a dna template from which a new dna strand can be copied and a pair of single stranded primers each with a 3- Oh group
what are the components of a PCR reaction
- dna strand for which a new strand can be copied
- pair of single stranded primers with OH group to
- solution that targets DNA, DNA polymerase and all 4 dNTPS
- magnesium ions and other salts that are necessary for the reaction
what is a primer
short fragments of dna
what are the 5 basic components of PCR
- dna template
- dna polymerase enzyme
- primers
- nucleotides
- reaction buffers
explain the steps of PCR cycle
Step 1: the dna is heated to 90-100 degrees to separate the dna strands
The dna is cooled to 30-65 so that the short single stranded primers can then anneal to their complimnetyar sequences
Step 3: the solution is heated to 72 degrees so dna plymerase can synthesize the new dna strands
Step 4: the cycle is repeated each tume the cycle is repeated the amount of target dna doubles
what is gene cloning
produce identical copies of a gene
- alter the DNA sequence
what is the process of gene cloning
main point: place gene into a host genome(bacteria) to be replicated, then the cloning vector carriers forhein gene into host cell
Dna fragment is inserted into a BACTERIAL cell and thecell is allowed to replicate the dna after the cell divides more of the dna fragement is then passed on to the daughter cell and then the cell is lysed to release their dna and the desired fragment is isolated from the rest of the bacterial dna
what is a cloning vector
stable replicating dna molecule to which foreign DNA fragment can be attached for introduction into a cell
what are the 3 elements of a cloning vector
Origin of replication;makes sure that the vector is replicated within the cell
Selectable markers: enable cells containing the vector to be selected or identified
Restriction sites into which a dna fragment can be inserted
define plasmid
Ciruclar dna molecules that exist naturally in bacteria- are commonly used as vectors for coling dna fragments in bacteria
how can a foreign dna fragment be inserted into a plasmid
Cut the foreign dna and the plasmid with the same restriction enzyme
-insert the DNA into the plasmid using ligase
- transfer the DNA into bacteria
If the restriction enzyme makes staggered cuts in the dna, complementary sticky ends are produced on the foreign dna and the plasmid
what is transformation in a plasmid
bacterial cells take up dna from the external environment
- Explain how to use selectable markers to identify recombinant cells (Fig 19.9)
- genes that have resistance are used as a selectable marker
- a plasmid that contains a fragment of lacz because it contains unique restriction sites into which if there is foreign DNA then B-galactosidase will be produced if not it wont produce it
- produce identical copies of a gene
in transformation only cells with ampR can grow where
on plates
if there is LacZ that produce b galactose present what color is it
blue
what color is it if there is lac z that doesn’t produce B galactose
white
what does expression of a foreign gene mean
- Describe how next-generation sequencing works, specifically Illumina sequencing (Fig 19.19)
Have made sequencing hundres of times faster and is less expensive than sanger sequencing
- sequencing in parallel
Illumnia sequencing
Most widely used sequencing
dNTPs that have a fluorecent tag are attatched
The dnTPs have a terminator group that once incorporated into the growing dna prevents the incorporation of any additional nucleotides
- Define DNA fingerprinting
uses dna for identification
what is microsatellites
tandem repreated sequence
number differs among individuals
short tandem repeats
- Describe how RNAi could be used to turn off a gene
- uses double stranded RNA (dsRNA) to target mRNA for degradation
- temporary turn off gene and observe phenotyp
fire and mellow experiment
- created transgenic worms that expression GFP
- ds unc22 RNA was injected and ds gfp was infected
how can we use RNAi to treat human disease
- choloestral ( targer apoB)
- cancer: target oncogene, multidrug resistance gene
- hiv: target viral genes
