lab techniques

Question 1

What are the steps of the polymerase chain reaction?

Answer 1

1) Denaturation-heat to generate 2 separate strands
2) Annealing-during cooling, excess premade DNA primers anneal to specific sequence on each strand to amplified
3) elongation-heat-stable DNA polymerase replicates DNA sequence following each primer

Question 2

what is the point of PCR?

Answer 2

amplify desired fragment of DNA

Question 3

What is used for size separation of PCR products?

Answer 3

agarose gel electrophoresis => smaller molecules travel further

Question 4

What is important about a southern blot?

Answer 4

DNA sample is exposed to DNA probe that anneals to its complementary strand=> double stranded, labeled DNA

Question 5

what is important for northern blot?

Answer 5

RNA sample used for studying mRNA levels

Question 6

what is important for western blot?

Answer 6

protein sample

Question 7

southwestern blot?

Answer 7

DNA-binding proteins (transcription factors) using labeled oligonucleotide probes

Question 8

define a lab microarray

Answer 8

1000s of nucleic acid sequences arranged on grids and DNA or RNA probes hybridized to chip and scanner detects relative amounts of complementary binding

Question 9

what is a microarray used for?

Answer 9

profile gene expression levels of 1000s of genes simultaneously;
detects SNPs

Question 10

what is a rapid immunologic technique testing for antigen-Ab reactivitiy?

Answer 10

enzyme-linked immunosorbent assay

Question 11

what does an INdirect ELISA test?

Answer 11

uses antigen test to see if specific Ab present in patient’s blood;
secondary Ab coupled to color generating enzyme added to detect 1st Ab

Question 12

What does a Direct ELISA test?

Answer 12

uses Ab coupled to color generating enzyme to see if specific antigen present in patient’s blood

Question 13

what happens if the ELISA test is positive?

Answer 13

test solution will have color change rxn

Question 14

What are ELISA tests typically used for?

Answer 14

determine whether Ab is present in patient’s blood sample

Question 15

Define FISH

Answer 15

fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes

Question 16

what is FISH used for?

Answer 16

specific localization of genes and direct visualization of anomalies (microdeletions) at molecular level => used for deletions when too small to karyotype

Question 17

what are the steps in cloning?

Answer 17

1) isolate eukaryotic mRNA (post processing)
2) expose mRNA to reverse transcriptase to produce cDNA
3) insert cDNA fragments into bacterial plasmids containing ABx resistance genes
4) surviving bacteria on ABx medium produce cDNA library

Question 18

what is the Cre-lox system associated w/ gene expression?

Answer 18

manipulate genes at specific developmental points using an ABx controlled promoter

Question 19

when would the cre-lox system be used?

Answer 19

to study a gene whose deletion causing embryonic death

Question 20

define RNA interference (RNAi)

Answer 20

dsRNA is synthesized that complementary to mRNA sequence of interest

Question 21

what happens when RNAi is transfected into human cells?

Answer 21

dsRNA separates and promotes degradation of target mRNA => knocks down gene expression

Question 22

define karyotyping

Answer 22

process that metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, banding pattern

Question 23

when can karyotyping be used?

Answer 23

performed on sample of blood, bone marrow, amniotic fluid, or placental tissue

Question 24

what can karyotyping be used to diagnose?

Answer 24

chromosomal imbalances (autosomal trisomies, sex chromosome disorders)